WO2017219350A1

WO2017219350A1 - Systems and methods for thermal cycling

Info

Publication number: WO2017219350A1
Application number: PCT/CN2016/087046
Authority: WO
Inventors: Xiang Li; Fei Yu
Original assignee: Coyote Bioscience Co Ltd
Current assignee: Coyote Bioscience Co Ltd
Priority date: 2016-06-24
Filing date: 2016-06-24
Publication date: 2017-12-28
Anticipated expiration: 2018-12-24
Also published as: CN109642197A

Abstract

Nucleic acid amplification systems and methods for performing nucleic acid amplifications with such systems are provided.

Description

SYSTEMS AND METHODS FOR THERMAL CYCLING

BACKGROUND

Nucleic acid amplification methods permit selected amplification and identification of nucleic acids of interest from a complex mixture, such as a biological sample. Nucleic acid of interest can be amplified via amplification methods known in the art, such as thermal cycling based approaches including polymerase chain reaction (PCR) . During or following amplification of the nucleic acid of interest, the products of amplification can be detected and results of the detection interpreted by an end user. Traditional nucleic acid amplification and detection methods typically involve a thermal cycling apparatus that requires a high voltage power input. Real-time PCR techniques involve the use of a detector that can detect a signal from a sample undergoing nucleic acid amplification in real-time. The combined thermal cycling and detection require a degree of power input that limits the use of the thermal cycler.

Point-of-care (POC) testing has the potential to improve the detection and management of infectious diseases in resource-limited settings with poor laboratory infrastructure, or in remote areas where there are delays in the receipt of laboratory results and potential complications to following up with patients. However, many challenges face performing nucleic acid amplification in POC settings. For instance, if a high voltage power input is needed to perform real-time PCR, the thermal cycler will have limited portability. For example, batteries may be quickly drained using such traditional systems. Similarly, the power sources that can be used to power such systems are limited, thus preventing full use of a thermal cycler in different environments and situations.

SUMMARY

A need exists for improved systems and methods for low power thermal cycling. Such low power thermal cycling may permit thermal cycling apparatuses to be portable and operable in different situations. For example, the thermal cycling apparatuses may be taken out into the field or into portions of the country where regular power sources are not readily available. The present disclosure provides systems and methods for performing thermal cycling rapidly and conveniently, making it possible to accommodate to different point-of-care (POC) settings.

An aspect of the present disclosure provides a system for nucleic acid amplification, comprising a base, a reaction vessel, a housing, a fluid flow member (or fluid flow unit) , and a controller.

In some embodiments, the base may comprise a heating member (or heating unit) . In some embodiments, the base may have a footprint that is less than or equal to about 5000 mm².

In some embodiments, the reaction vessel may comprise a reaction chamber. In some embodiments, the reaction member may be adjacent to and in thermal communication with the heating member. In some embodiments, during use, the heating member may provide thermal energy to a reaction mixture in the reaction chamber. In some embodiments, the reaction mixture may comprise a nucleic acid sample and reagents necessary for nucleic acid amplification.

In some embodiments, the housing may be removably mountable to the base. In some embodiments, the housing may encapsulate the reaction vessel when mounted to the base. In some embodiments, the housing may include at least one outlet that permits flow of a convective fluid from a source of the convective fluid across the reaction chamber to the at least one outlet.

In some embodiments, the fluid flow member may subject the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet.

In some embodiments, the controller may be operatively coupled to the heating member and the fluid flow member. In some embodiments, the controller may be programmed to subject the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture. In some embodiments, subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture may be performed by (i) directing the heating member to provide thermal energy to the reaction chamber, and (ii) directing the fluid flow member to subject the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet.

In some embodiments, the reaction chamber may have a footprint that is less than or equal to about 1000 mm².

In some embodiments, the footprint may be less than or equal to about 500 mm².

In some embodiments, the footprint may be less than or equal to about 300 mm².

In some embodiments, the base may have a footprint that is less than or equal to about 2000 mm².

In some embodiments, the housing mounted to the base may have a footprint that is less than or equal to about 5000 mm².

In some embodiments, a cross-section of the reaction chamber may be less than a cross-section of the heating member.

In some embodiments, the reaction chamber may have a surface area to volume ratio of at least 100 mm^-1.

In some embodiments, the system may further comprise a power supply that supplies power to the heating member.

In some embodiments, the power supply may supply power to the fluid flow member.

In some embodiments, the system may further comprise a switch in the base or the housing operatively connected to the power supply. In some embodiments, the switch may regulate supply of power from the power supply to the heating member.

In some embodiments, the switch may direct supply of power from the power supply to the heating member when the housing is mounted to the base.

In some embodiments, the housing may comprise a chamber that encapsulates the reaction vessel when mounted to the base.

In some embodiments, the system may further comprise a sample holder mounted to the heating member. In some embodiments, the sample holder may receive and secure the reaction vessel.

In some embodiments, the reaction chamber may be adjacent to and in thermal communication with the heating member when the reaction vessel is secured to the sample holder.

In some embodiments, the base may further comprise an excitation energy source operatively coupled to the reaction vessel. In some embodiments, the excitation energy source may provide excitation energy to the reaction vessel to induce a signal (s) that is indicative of a presence or absence of an amplification product of the nucleic acid amplification reaction.

In some embodiments, the system may further comprise a first sensor in sensing communication with the reaction vessel. In some embodiments, the first sensor may detect the signal (s) that is indicative of the presence or absence of the amplification product.

In some embodiments, the first sensor may be an optical sensor, and the signal (s) may be an optical signal.

In some embodiments, the system may further comprise a second sensor in sensing communication with the reaction vessel. In some embodiments, the second sensor may detect a temperature at one or more location (s) within the reaction chamber of the reaction vessel.

In some embodiments, the system may further comprise a display in the base or the housing operatively coupled to the first sensor. In some embodiments, the display may be configured to display the signal (s) indicative of the presence or absence of the amplification product.

In some embodiments, the system may further comprise a display in the base or the housing operatively coupled to the first sensor and/or the second sensor. In some embodiments, the display may be configured to 1) display the signal (s) indicative of the presence or absence of the amplification product, and/or 2) display the temperature at the one or more location (s) within the reaction chamber of the reaction vessel.

In some embodiments, the excitation energy source may be a light source.

In some embodiments, the convective fluid may be a convective gas.

In some embodiments, the convective gas may be air.

In some embodiments, the fluid flow member may be a fan.

In some embodiments, the fluid flow member may be mounted to the housing or to the base.

In some embodiments, the source of the convective fluid may be a refrigeration unit.

In some embodiments, the heating member may include an infrared heating unit.

In some embodiments, the heating member may include a Peltier heating unit.

In some embodiments, the heating member may include an aluminum-containing heating unit.

In some embodiments, the heating member may include an electrically resistive heating unit.

In some embodiments, the controller may be programmed to direct the heating member to provide thermal energy to the reaction chamber until the reaction mixture reaches a first temperature, and direct the fluid flow member to subject the convective fluid to flow across the reaction chamber until the reaction mixture reaches a second temperature that is less than the first temperature.

In some embodiments, the controller may be programmed to direct the fluid flow member to reduce or terminate flow of the convective fluid when the reaction mixture reaches the second temperature.

In some embodiments, the controller may be included in the base.

In some embodiments, the reaction mixture may include one or more primers and polymerizing enzymes.

In some embodiments, the reaction mixture may include a buffer.

In some embodiments, the reaction mixture may include cations that regulate an activity of the polymerizing enzymes.

In some embodiments, the cations may include Mg²⁺ or Mn²⁺.

In some embodiments, the one or more primers may have nucleic acid sequences that are selected for HBV, HCV, FluA, FluB, CA16, EV71, enterovirus, EBOV, EBV, measles virus, salmonella, HPV and/or HIV.

In some embodiments, the nucleic acid amplification reaction may be polymerase chain reaction (PCR) .

In some embodiments, the convective fluid may be at an average temperature of less than about 15℃.

In some embodiments, the convective fluid may be at an average temperature of less than about 10℃.

In some embodiments, the controller may provide heating and/or cooling to the reaction mixture by controlling a heating rate of the reaction chamber using the thermal energy provided by the heating member and a cooling rate of the reaction chamber using the flow of the convective fluid across the reaction chamber.

In some embodiments, the reaction mixture may be subjected to heating when the heating rate is greater than the cooling rate.

In some embodiments, the reaction mixture may be subjected to cooling when the heating rate is less than the cooling rate.

In some embodiments, the heating rate may be at least 5℃/s.

In some embodiments, the cooling rate may be at least 5℃/s.

In some embodiments, the controller may not subject the reaction mixture to the one or more cycles of heating and cooling in the absence of the switch being turned on.

In some embodiments, the controller may subject the reaction mixture to the one or more cycles of heating and cooling after a time delay upon the switch being turned on.

In some embodiments, the reaction vessel may further comprise a sampling unit. In some embodiments, the sampling unit may comprise a sampling chamber in fluid communication with the reaction chamber.

In some embodiments, the sampling unit may further comprise a collection member that collects a nucleic acid sample.

In some embodiments, the sampling unit may further comprise a sealing member that seals an opening of the sampling chamber.

In some embodiments, during use, the collection member may pierce the sealing member to release the nucleic acid sample into the sampling chamber.

In some embodiments, a side of the reaction chamber adjacent to the heating member may have a thickness of less than about 1 mm.

An aspect of the present disclosure provides a method for nucleic acid amplification, comprising:

activating a system comprising (i) a base, (ii) a reaction vessel, (iii) a housing, and (iv) a fluid flow member； and

subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture.

In some embodiments, the base may comprise a heating member. In some embodiments, the base may have a footprint that is less than or equal to about 5000 mm².

In some embodiments, the reaction vessel may comprise a reaction chamber. In some embodiments, the reaction member may be adjacent to and in thermal communication with the heating member. In some embodiments, the reaction chamber may comprise a reaction mixture comprising a nucleic acid sample and reagents necessary for nucleic acid amplification.

In some embodiments, the housing may be removably mountable to the base. In some embodiments, the housing may encapsulate the reaction vessel. In some embodiments, the housing may include at least one outlet that permits flow of a convective fluid from a source of the convective fluid across the reaction chamber to the at least one outlet.

In some embodiments, subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture may be performed by (i) directing the heating member to provide thermal energy to the reaction chamber, and (ii) directing the fluid flow member to subject the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet.

In some embodiments, the housing may be mounted to the base has a footprint that is less than or equal to about 5000 mm².

In some embodiments, the activating in (a) may be performed by supplying power to the heating member with a power supply.

In some embodiments, the power may be supplied by turning on a switch operatively connected to the power supply in the base or the housing.

In some embodiments, the switch may be turned on by mounting the housing to the base.

In some embodiments, the method may further comprise, prior to (a) , securing the reaction vessel to a sample holder mounted to the heating member.

In some embodiments, the method may further comprise providing excitation energy to the reaction vessel to induce a signal (s) that is indicative of a presence or absence of an amplification product of the nucleic acid amplification reaction.

In some embodiments, the method may further comprise providing a first sensor in sensing communication with the reaction vessel to detect the signal (s) that is indicative of the presence or absence of the amplification product.

In some embodiments, the method may further comprise providing a second sensor in sensing communication with the reaction vessel to detect a temperature at one or more location (s) within the reaction chamber of the reaction vessel.

In some embodiments, the method may further comprise providing a display operatively coupled to the first sensor and/or the second sensor in the base or the housing. In some embodiments, the display may be configured to: (1) display the signal (s) indicative of the presence or absence of the amplification product, and/or (2) display the temperature at the one or more location (s) within the reaction chamber of the reaction vessel.

In some embodiments, the method may further comprise providing a display operatively coupled to the first sensor in the base or the housing. In some embodiments, the display may be configured to display the signal (s) indicative of the presence or absence of the amplification product.

In some embodiments, the excitation energy source may be a light source.

In some embodiments, the convective fluid may be a convective gas.

In some embodiments, the convective gas may be air.

In some embodiments, the fluid flow member may be a fan.

In some embodiments, the method may further comprise, prior to (a) , depositing the base having the housing mounted thereto in a refrigeration unit.

In some embodiments, the heating member may include an infrared heating unit.

In some embodiments, the heating member may include a Peltier heating unit.

In some embodiments, thermal energy may be provided to the reaction chamber until the reaction mixture reaches a first temperature. In some embodiments, the convective fluid may be subjected to flow across the reaction chamber until the reaction mixture reaches a second temperature that is less than the first temperature.

In some embodiments, the method may further comprise reducing or terminating flow of the convective fluid when the reaction mixture reaches the second temperature.

In some embodiments, the reaction mixture may include a buffer.

In some embodiments, the cations may include Mg²⁺ or Mn²⁺.

In some embodiments, the method may further comprise controlling a heating rate of the reaction chamber using the thermal energy provided by the heating member and a cooling rate of the reaction chamber using the flow of the convective fluid across the reaction chamber.

In some embodiments, the heating rate may be greater than the cooling rate, thereby subjecting the reaction mixture to heating.

In some embodiments, the heating rate may be less than the cooling rate, thereby subjecting the reaction mixture to cooling.

In some embodiments, the heating rate may be at least 5℃/s.

In some embodiments, the cooling rate may be at least 5℃/s.

In some embodiments, in (b) , the reaction mixture may not be subjected to the one or more cycles of heating and cooling in the absence of the switch being turned on.

In some embodiments, in (b) , the reaction mixture may not be subjected to the one or more cycles of heating and cooling after a time delay upon the switch being turned on.

In some embodiments, the method may further comprise, subsequent to (b) , deactivating the system.

In some embodiments, the method may further comprise, prior to (a) , depositing the nucleic acid sample in the reaction vessel.

In some embodiments, the reaction vessel may further comprise a sampling unit, and the sampling unit comprises a collection member and a sampling chamber in fluid communication with the reaction chamber. In some embodiments, the nucleic acid sample may be deposited in the reaction vessel by piercing a sealing member sealing an opening of the sampling chamber with the collection member having collected the nucleic acid sample thereon or therein.

An aspect of the present disclosure provides a system for nucleic acid amplification, comprising: a base, a reaction vessel, a housing, and a controller.

In some embodiments, the housing may be removably mountable to the base. In some embodiments, the housing may encapsulate the reaction vessel. In some embodiments, the housing may include a cooling member that is in thermal communication with the reaction vessel when the housing is mounted to the base.

In some embodiments, the controller may be coupled to the heating member. In some embodiments, the controller may be programmed to subject the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture. In some embodiments, subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture may be performed by regulating (i) a rate of heating of the reaction vessel using the heating member and (ii) a rate of cooling of the reaction vessel using the cooling member.

In some embodiments, the cooling member may be formed of a material with a heat capacity of at least about 0.2 J /g*K.

In some embodiments, the cooling member may be formed of a material with a heat capacity of at least about 0.3 J /g*K.

In some embodiments, the cooling member may be formed of a material with a heat capacity of at least about 0.4 J /g*K.

In some embodiments, the cooling member may be formed of a material with a heat capacity of at least about 0.5 J /g*K.

In some embodiments, the cooling member may be formed of a material with a heat capacity of at least about 1.0 J /g*K.

In some embodiments, the cooling member is a solid comprising copper.

activating a system comprising (i) a base, (ii) a reaction vessel, and (iii) a housing； and

In some embodiments, subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture may be performed by regulating (i) a rate of heating of the reaction vessel using the heating member and (ii) a rate of cooling of the reaction vessel using the cooling member.

In some embodiments, the method may further comprise providing a display operatively coupled to the first sensor and/or the second sensor in the base or the housing. In some embodiments, the display is configured to: (1) display the signal (s) indicative of the presence or absence of the amplification product, and/or (2) display the temperature at the one or more location (s) within the reaction chamber of the reaction vessel.

In some embodiments, the method may further comprise providing a display operatively coupled to the first sensor in the base or the housing. In some embodiments, the display is configured to display the signal (s) indicative of the presence or absence of the amplification product.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1A and 1B demonstrate an example of a system of the present disclosure.

FIG. 2 demonstrates an example of a system of the present disclosure.

FIG. 3 demonstrates internal structure of a system of the present disclosure.

FIG. 4 demonstrates internal structure of a system of the present disclosure.

FIG. 5 demonstrates a display that may be comprised in a system of the present disclosure.

FIG. 6 demonstrates a reaction vessel of the present disclosure.

FIG. 7 shows a cross-sectional view of a reaction vessel of the present disclosure.

FIG. 8 shows a bottom view of a reaction vessel of the present disclosure.

FIG. 9 shows a computer control system that is programmed or otherwise configured to implement a method of the present disclosure.

DETAILED DESCRIPTION

While preferable embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

As used herein, the term “sample” generally refers to any sample containing or suspected of containing a nucleic acid molecule. For example, a subject sample may be a biological sample containing one or more nucleic acid molecules. The biological sample may be obtained (e.g., extracted or isolated) from a bodily sample of a subject that may be selected from blood (e.g., whole blood) , plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears. The bodily sample may be a fluid or tissue sample (e.g., skin sample) of the subject. In some examples, the sample is obtained from a cell-free bodily fluid of the subject, such as whole blood. In such instance, the sample can include cell-free DNA and/or cell-free RNA. In some other examples, the sample is an environmental sample (e.g., soil, waste, ambient air and etc. ) , industrial sample (e.g., samples from any industrial processes) , and food samples (e.g., dairy products, vegetable products, and meat products) .

In some embodiments, a sample is obtained directly from a subject without further processing. In some embodiments, a sample is processed prior to a biological or chemical reaction (e.g., nucleic acid amplification) . For example, a lysis agent may be added to a sample holder prior to adding a biological sample and reagents necessary for nucleic acid amplification. Examples of the lysis agent include Tris-HCl, EDTA, detergents (e.g., Triton X-100, SDS) , lysozyme, glucolase, proteinase E, viral endolysins, exolysins, zymolyase, lyticase, proteinase K, endolysins and exolysins from bacteriophages, endolysins from bacteriophage PM2, endolysins from the B. subtilis bacteriophage PBSX, endolysins from Lactobacillus prophages Lj928, Lj965, bacteriophage 15 Phiadh, endolysin from the Streptococcus pneumoniae bacteriophage Cp-I, bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30, endolysins and exolysins from prophage bacteria, endolysins from Listeria bacteriophages, holin-endolysin, cell 20 lysis genes, holWMY Staphylococcus wameri M phage varphiWMY, Iy5WMY of the Staphylococcus wameri M phage varphiWMY, Tween 20, PEG, KOH, NaCl, and combinations thereof. In some embodiments, a lysis agent is sodium hydroxide (NaOH) . In some embodiments, the biological sample is not treated with a detergent.

In some embodiments, the sample is purified (e.g., by filtration, centrifugation, column purification and/or magnetic purification, for example, by using magnetic beads (e.g., super paramagnetic beads) ) to obtain purified nucleic acids.

A sample may be of any suitable size or volume. In some examples, a small volume comprises no more than about 5 mL； no more than about 4 mL； no more than about 3 mL； no more than about 2 mL； no more than about 1 mL； no more than about 500 μL； no more than about 250 μL； no more than about 100 μL； no more than about 90 μL； no more than about 80 μL； no more than about 70 μL； no more than about 60 μL； no more than about 50 μL； no more than about 40 μL； no more than about 30 μL； no more than about 25 μL； no more than about 20 μL； no more than about 15 μL； no more than about 10 μL； no more than about 8 μL； no more than about 6 μL； no more than about 5 μL； no more than about 4 μL； no more than about 3 μL； no more than about 2 μL； no more than about 1 μL； no more than about 0.8 μL； no more than about 0.5 μL； no more than about 0.3 μL； no more than about 0.2 μL； no more than about 0.1 μL； no more than about 0.05 μL； or no more than about 0.01 μL.

As used herein, the term “bodily fluid” generally refers to any fluid obtainable from a subject. A bodily fluid may include but not limited to, e.g. blood, urine, saliva, tears, sweat, a bodily secretion, a bodily excretion, or any other fluid originating in or obtainable from a subject. In particular, bodily fluids include but not limited to blood, serum, plasma, bone marrow, saliva, urine, gastric fluid, spinal fluid, tears, stool, mucus, sweat, earwax, oil, glandular secretions, cerebral spinal fluid, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, meconium, breast milk and/or other secretions or excretions.

As used herein, the term “nucleic acid” generally refers to a molecule comprising one or more nucleic acid subunits. A nucleic acid may include one or more subunits selected from adenosine (A) , cytosine (C) , guanine (G) , thymine (T) and uracil (U) , or variants thereof. A nucleotide can include A, C, G, T or U, or variants thereof including but not limited to peptide nucleic acid (PNA) . A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C, T or U, or variant thereof) . A subunit can enable individual nucleic acid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or uracil-counterparts thereof) to be resolved. In some examples, a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) , or derivatives thereof. A nucleic acid may be single-stranded or double stranded. A nucleic acid may comprise one or more modified nucleotides, e.g., methylated nucleotides and nucleotide analogs.

As used herein, the term “polymerase” generally refers to any enzyme capable of catalyzing a polymerization reaction. Examples of polymerases include e.g., a nucleic acid polymerase, a transcriptase or a ligase. A polymerase can be a polymerization enzyme or a polymerizing enzyme.

As used herein, the term “subject” generally refers to an entity or a medium that has testable or detectable genetic information. A subject may be a person or individual. A subject may be a vertebrate, such as, for example, a mammal. Examples of subjects include murines, simians, humans, farm animals, sport animals, pets, avians, canines, felines, equines, bovines, ovines, porcines, dolphins, rodents (e.g., mice, rats) , or insects. Other examples of subjects include, for example, food, plant, soil, and water. A subject may be a living subject or a dead subject. The subject may be a human or an animal.

As used herein, the term “about” or “nearly” generally refers to a reasonable variation, e.g. within +/-10％, 9％, 8％, 7％, 6％, 5％, 4％, 3％, 2％, or 1％of a designated amount.

As used herein, the term “reaction mixture” generally refers to a composition comprising reagents necessary to complete nucleic acid amplification (e.g., DNA amplification, RNA amplification) , with non-limiting examples of such reagents that include primer sets having specificity for target RNA or target DNA, DNA produced from reverse transcription of RNA, a DNA polymerase, a reverse transcriptase (e.g., for reverse transcription of RNA) , suitable buffers (including zwitterionic buffers) , co-factors (e.g., divalent and monovalent cations) , dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG) ) , etc) . In some cases, reaction mixtures can also comprise one or more reporter agents.

As used herein, a “reporter agent” generally refers to a composition that yields a detectable signal, the presence or absence of which can be used to detect the presence of amplified product.

As used herein, the term “target nucleic acid” generally refers to a nucleic acid molecule in a starting population of nucleic acid molecules having a nucleotide sequence whose presence, amount, and/or sequence, or changes in one or more of these, are desired to be determined. A target nucleic acid may be any type of nucleic acid, including DNA, RNA, and analogues thereof. As used herein, a “target ribonucleic acid (RNA) ” generally refers to a target nucleic acid that is RNA. As used herein, a “target deoxyribonucleic acid (DNA) ” generally refers to a target nucleic acid that is DNA.

In one aspect, the present disclosure provides a system for nucleic acid amplification. The system may comprise a base, the base may comprise a heating member, and the base may have a footprint that is less than or equal to about 9000 mm². For example, the base may have a footprint that is less than or equal to about 8000 mm², about 7000 mm², about 6000 mm², about 5000 mm², about 4500 mm², about 4000 mm², about 3500 mm², about 3000 mm², about 2500 mm², about 2000 mm², about 1500 mm², about 1000 mm², about 900 mm², about 800 mm², about 700 mm², about 600 mm², about 500 mm², about 400 mm², about 300 mm², about 200 mm², about 100 mm², etc.

The system may further comprise a reaction vessel comprising a reaction chamber. The reaction chamber may be adjacent to and in thermal communication with the heating member. During use, the heating member may provide thermal energy to a reaction mixture in the reaction chamber. The reaction mixture may comprise a nucleic acid sample and reagents necessary for nucleic acid amplification. The reaction chamber may have a footprint that is less than or equal to about 2000 mm². For example, the reaction chamber may have a footprint that is less than or equal to about 1500 mm², about 1000 mm², about 900 mm², about 800 mm², about 700 mm², about 600 mm², about 500 mm², about 400 mm², about 300 mm², about 200 mm², about 100 mm², etc. In some embodiments, a cross-section of the reaction chamber is less than a cross-section of the heating member.

In another aspect, the present disclosure provides a method for nucleic acid amplification. The method may comprise activating a system comprising (i) a base comprising a heating member, wherein the base has a footprint that is less than or equal to about 5000 mm², (ii) a reaction vessel comprising a reaction chamber, wherein the reaction chamber is adjacent to and in thermal communication with the heating member. The reaction chamber may comprise a reaction mixture comprising a nucleic acid sample and reagents necessary for nucleic acid amplification. The system may further comprise (iii) a housing removably mounted to the base. The housing may encapsulate the reaction vessel, and the housing may include at least one outlet that permits flow of a convective fluid from a source of the convective fluid across the reaction chamber to the at least one outlet. The system may also comprise (iv) a fluid flow member that subjects the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet. The method may further comprise subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture, which may be performed by (i) directing the heating member to provide thermal energy to the reaction chamber, and (ii) directing the fluid flow member to subject the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet.

In some embodiments, the base may have a footprint that is less than or equal to about 9000 mm². For example, the base may have a footprint that is less than or equal to about 8000 mm², about 7000 mm², about 6000 mm², about 5000 mm², about 4500 mm², about 4000 mm², about 3500 mm², about 3000 mm², about 2500 mm², about 2000 mm², about 1500 mm², about 1000 mm², about 900 mm², about 800 mm², about 700 mm², about 600 mm², about 500 mm², about 400 mm², about 300 mm², about 200 mm², about 100 mm², etc.

In any of the various aspects, the housing and/or the base may have more than one outlets, for example, the housing and/or the base may have 2, 3, 4, 5, 6, 7, 8, 9 or more outlets that permit flow of a convective fluid from a source of the convective fluid across the reaction chamber to the at least one outlet.

The reaction vessel may be any container capable of receiving a solution comprising a nucleic acid sample (e.g., a PCR tube) . The reaction vessel may comprise a reaction chamber and chemical and/or biological reactions may occur in the reaction chamber. The reaction vessel may be of varied size, shape, weight, and configuration. In some embodiments, the reaction vessel is round or oval tubular shaped. In some embodiments, the reaction vessel is rectangular, square, diamond, circular, elliptical, or triangular shaped. The reaction vessel may be regularly shaped or irregularly shaped. For example, a reaction vessel may be a tube, a well, a capillary tube, a cartridge, a cuvette, a centrifuge tube, or a pipette tip. In some embodiments, the reaction vessel has a surface area to volume ratio of at least 100 mm^-1, 200 mm^-1, 300 mm^-1, 350 mm^-1, 400 mm^-1, 450 mm^-1, 500 mm^-1 or more. A side of the reaction chamber adjacent to the heating member may have a thickness of less than about 3 mm. For example, a side of the reaction chamber adjacent to the heating member may have a thickness of less than about 3 mm, 2.5 mm, 2.0 mm, 1.5 mm, 1.0 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.05 mm, etc.

The reaction vessel may comprise a sampling unit. The sampling unit may comprise a sampling chamber in fluid communication with the reaction chamber. The sampling unit may comprise a collection member that collects a nucleic acid sample. In some embodiments, the sampling unit comprises a sealing member that seals an opening of the sampling chamber. During use, the collection member may pierce the sealing member to release the nucleic acid sample into the sampling chamber.

The sealing member may be any suitable structure that separates at least two volumes, or that separates a volume from an external environment. A sealing member may be a synthetic membrane, e.g., a membrane formed of a solid state material (e.g., semiconductor, metal, semi-metal or non-metal) or polymeric material (e.g., a polymeric membrane) . For example, a sealing member can be formed by an opaque, transparent, or translucent material sealing a sampling chamber and separating it from the external environment. In some embodiments, the sealing member is a polymeric membrane made from parafilm.

The system may comprise a housing removably mountable to the base. The housing may encapsulate the reaction vessel when mounted to the base, and the housing may include at least one outlet that permits flow of a convective fluid from a source of the convective fluid across the reaction chamber to the at least one outlet. In some embodiments, the housing comprises more than one outlet, e.g., 2, 3, 4, 5, 6, 7, or more outlets. The housing may have a footprint that is less than or equal to about 9000 mm². For example, the housing may have a footprint that is less than or equal to about 8000 mm², about 7000 mm², about 6000 mm², about 5000 mm², about 4500 mm², about 4000 mm², about 3500 mm², about 3000 mm², about 2500 mm², about 2000 mm², about 1500 mm², about 1000 mm², about 900 mm², about 800 mm², about 700 mm², about 600 mm², about 500 mm², about 400 mm², about 300 mm², about 200 mm², about 100 mm², etc. The housing may comprise a chamber that encapsulates the reaction vessel when mounted to the base. In some embodiments, the housing comprises a chamber that at least partially encapsulates the reaction vessel when mounted to the base.

In addition, the system may comprise a fluid flow member that subjects the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet. The convective fluid may be a convective gas, such as air, or a mixture of one or more gases. The source of the convective fluid may be a refrigeration unit, such as a refrigerator. The fluid flow member may be a fan. In some embodiments, the fluid flow member is mounted to the housing or to the base.

The system may further comprise a controller operatively coupled to the heating member and the fluid flow member. The controller may be programmed to subject the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture. This may be performed by e.g., (i) directing the heating member to provide thermal energy to the reaction chamber, and (ii) directing the fluid flow member to subject the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet. The controller may be included in the base.

The system may further comprise a power supply. The power supply may be configured to supply power to the heating member, and/or the fluid flow member. Activating the system may be achieved by supplying power to the heating member and/or the fluid flow member with a power supply.

The system may comprise a switch in the base and/or in the housing. The switch may be operatively connected to the power supply, and may regulate supply of power from the power supply to the heating member and/or the fluid flow member. For example, the switch may direct supply of power from the power supply to the heating member and/or the fluid flow member when the housing is mounted to the base. Power may be supplied by turning on the switch operatively connected to the power supply in the base or the housing. The switch may be turned on by mounting the housing to the base.

The system may comprise a sample holder. The sample holder may receive and secure the reaction vessel. The reaction chamber of a reaction vessel may be adjacent to and in thermal communication with the heating member when the reaction vessel is secured to the sample holder. In some embodiments, the sample holder is mounted to the heating member； this may ensure that the reaction vessel or the sample therein is in thermal communication with the heating member when secured in or on the sample holder.

In a system of the present disclosure, the base may further comprise an excitation energy source operatively coupled to the reaction vessel. The excitation energy source may provide excitation energy to the reaction vessel to induce a signal (s) that is indicative of a presence or absence of an amplification product of the nucleic acid amplification reaction. The excitation energy source may be a light source, or other inductive energy sources, e.g., a light-emitting diode, a laser or other energy sources. The excitation energy source may be activated to direct excitation energy to the sample comprised in the reaction chamber, thereby generating emitted signals (e.g., optical signals, fluorescent signals and/or electrostatic signals) indicating occurrence and/or result of the chemical or biological reaction (e.g., nucleic acid amplification reaction) on the sample.

The system may comprise one or more sensors, e.g., 2, 3, 4, 5, 6, 7 or more sensors. For example, the system may comprise a first sensor in sensing communication with the reaction vessel. The first sensor may detect the signal (s) indicative of a presence or absence of an amplification product. The first sensor may be an optical sensor, an inductive sensor, an electrochemical sensor, an electrostatic sensor, and/or an impedance sensor. The signal (s) may be an optical signal, a fluorescent signal and/or an electrostatic signal.

The system may further comprise a second sensor in sensing communication with the reaction vessel. The second sensor may detect a temperature at one or more location (s) within the reaction chamber of the reaction vessel.

The system may further comprise one or more (e.g., 1, 2, 3, or more) display (s) in the base and/or the housing operatively coupled to the first sensor and/or the second sensor. The display may be configured to display the signal (s) indicative of the presence or absence of the amplification product, and/or the temperature at the one or more location (s) within the reaction chamber of the reaction vessel.

The heating member may include an infrared heating unit, a Peltier heating unit, an aluminum-containing heating unit, and/or an electrically resistive heating unit.

The controller may be programmed to direct the heating member to provide thermal energy to the reaction chamber until the reaction mixture reaches a first temperature, and direct the fluid flow member to subject the convective fluid to flow across the reaction chamber until the reaction mixture reaches a second temperature that is less than the first temperature. The controller may be programmed to direct the fluid flow member to reduce or terminate flow of the convective fluid when the reaction mixture reaches the second temperature.

The first temperature may be from about 80 ℃ to about 100 ℃. For example, the first temperature may be from about 87 ℃ to about 95 ℃, or from about 90 ℃ to about 95 ℃. The first temperature may be from about 92 ℃ to about 95 ℃. The first temperature may be greater than or equal to about 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 ℃, 98 ℃, 99 ℃, or 100 ℃. The second temperature may be from about 40 ℃ to about 70 ℃, or from about 50 ℃ to about 60 ℃. The second temperature may be less than or equal to about 40 ℃, 45 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, or 85 ℃.

The reaction mixture may include one or more primers and polymerizing enzymes. In some embodiments, the reaction mixture includes a buffer. The reaction mixture may include cations that regulate an activity of the polymerizing enzymes. The cations may include Mg²⁺ and/or Mn²⁺. The one or more primers may have sequences complementary with a target nucleic acid sequence.

In any of the various aspects, primers sets directed to a target nucleic acid may be utilized to conduct nucleic acid amplification reaction. Primer sets generally comprise one or more primers. For example, a primer set may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more primers. In some embodiments, a primer set comprises primers directed to different amplified products or different nucleic acid amplification reactions. For example, a primer set may comprise a first primer necessary to generate a first strand of nucleic acid product that is complementary to at least a portion of the target nucleic acid and a second primer complementary to the nucleic acid strand product necessary to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product.

For example, a primer set may be directed to a target RNA. The primer set may comprise a first primer that can be used to generate a first strand of nucleic acid product that is complementary to at least a portion the target RNA. In the case of a reverse transcription reaction, the first strand of nucleic acid product may be DNA. The primer set may also comprise a second primer that can be used to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product. In the case of a reverse transcription reaction conducted in parallel with DNA amplification, the second strand of nucleic acid product may be a strand of nucleic acid (e.g., DNA) product that is complementary to a strand of DNA generated from an RNA template.

Where desired, any suitable number of primer sets may be used. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primer sets may be used. Where multiple primer sets are used, one or more primer sets may each correspond to a particular nucleic acid amplification reaction or amplified product.

In some embodiments, a DNA polymerase is used. Any suitable DNA polymerase may be used, including commercially available DNA polymerases. A DNA polymerase generally refers to an enzyme that is capable of incorporating nucleotides to a strand of DNA in a template bound fashion. Non-limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, and variants, modified products and derivatives thereof. For certain Hot Start Polymerase, a denaturation step at a temperature from about 92℃ to 95℃ (e.g., 94℃ to 95℃) for a time period from about 2 minutes to 10 minutes may be required, which may change the thermal profile based on different polymerases.

In some embodiments, a reverse transcriptase is used. Any suitable reverse transcriptase may be used. A reverse transcriptase generally refers to an enzyme that is capable of incorporating nucleotides to a strand of DNA, when bound to an RNA template. Non-limiting examples of reverse transcriptases include HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and variants, modified products and derivatives thereof.

The target nucleic acid sequence may be associated with a disease. The disease may be associated with a virus such as for example an RNA virus or a DNA virus. In some embodiments, the virus can be selected from the group consisting of human immunodeficiency virus I (HIV I) , human immunodeficiency virus II (HIV II) , an orthomyxovirus, Ebola virus, Dengue virus, influenza viruses, hepevirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus, and Varicella virus. In some embodiments, the influenza virus is selected from the group consisting of H1N1 virus, H3N2 virus, H7N9 virus and H5N1 virus. In some embodiments, the adenovirus is adenovirus type 55 (ADV55) or adenovirus type 7 (ADV7) . In some embodiments, the hepatitis C virus is armored RNA-hepatitis C virus (RNA-HCV) . In some embodiments, the disease is associated with a pathogenic bacterium (e.g., Mycobacterium tuberculosis) or a pathogenic protozoan (e.g., Plasmodium) .

In some embodiments, the disease is cancer. Non-limiting examples of the cancers include colorectal cancer, bladder cancer, ovarian cancer, testicular cancer, breast cancer, skin cancer, lung cancer, pancreatic cancer, stomach cancer, esophageal cancer, brain cancer, leukemia, liver cancer, endometrial cancer, prostate cancer, and head and neck cancer.

In some embodiments, the one or more primers have nucleic acid sequences that are selected for HBV, HCV, FluA, FluB, CA16, EV71, enterovirus, EBOV, EBV, measles virus, salmonella, HPV and/or HIV.

A variety of nucleic acid amplification reactions may be used to amplify a target nucleic acid in the nucleic acid sample and generate an amplified product. Moreover, amplification of a nucleic acid may be linear, exponential, or a combination thereof. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction, ligase chain reaction, helicase-dependent amplification (e.g., amplification that is preceded by contacting the nucleic acid with a helicase) , asymmetric amplification, rolling circle amplification, and multiple displacement amplification (MDA) . In some embodiments, the amplified product may be DNA. In cases where a target RNA is amplified, DNA can be obtained by reverse transcription of the RNA and subsequent amplification of the DNA can be used to generate an amplified DNA product. The amplified DNA product may be indicative of the presence of the target RNA in the biological sample. In cases where DNA is amplified, any DNA amplification method known in the art may be employed. Non-limiting examples of DNA amplification methods include polymerase chain reaction (PCR) , variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric interlaced PCR, touchdown PCR) , and ligase chain reaction (LCR) . In some embodiments, DNA amplification is linear. In some embodiments, DNA amplification is exponential. In some embodiments, DNA amplification is achieved with nested PCR, which can improve sensitivity of detecting amplified DNA products.

In any of the various aspects, nucleic acid amplification reactions described herein may be conducted in parallel. In general, parallel amplification reactions are amplification reactions that occur in the same reaction chamber and at the same time. Parallel nucleic acid amplification reactions may be conducted, for example, by including reagents necessary for each nucleic acid amplification reaction in a reaction chamber to obtain a reaction mixture and subjecting the reaction mixture to conditions necessary for each nucleic amplification reaction. For example, reverse transcription amplification and DNA amplification may be conducted in parallel, by providing reagents necessary for both amplification methods in a reaction chamber to obtain a reaction mixture and subjecting the reaction mixture to conditions suitable for conducting both amplification reactions. DNA generated from reverse transcription of the RNA may be amplified in parallel to generate an amplified DNA product. Any suitable number of nucleic acid amplification reactions may be conducted in parallel. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 10,000, or more nucleic acid amplification reactions are conducted in parallel.

The convective fluid may be at an average temperature of less than about 25℃. For example, the convective fluid may be at an average temperature of less than about 24℃, 23℃, 22℃, 21℃, 20℃, 19℃, 18℃, 17℃, 16℃, 15℃, 14℃, 13℃, 12℃, 11℃, 10℃, 9℃, 8℃, 7℃, 6℃, 5℃, 4℃, 3℃, 2℃, 1℃, 0℃.

The controller may provide heating and/or cooling to the reaction mixture by controlling a heating rate of the reaction chamber using the thermal energy provided by the heating member and a cooling rate of the reaction chamber using the flow of the convective fluid across the reaction chamber. The reaction mixture may be subjected to heating when the heating rate is greater than the cooling rate. The reaction mixture may be subjected to cooling when the heating rate is less than the cooling rate. The heating rate may be at least 0.1℃/s. For example, the heating rate may be at least 0.5℃/s, 1℃/s, 2℃/s, 3℃/s, 4℃/s, 5℃/s, 6℃/s, 7℃/s, 8℃/s, 9℃/s, 10℃/s, 11℃/s, 12℃/s, 13℃/s, 14℃/s, 15℃/s, 16℃/s, 17℃/s, 18℃/s, 19℃/s, 20℃/s, 21℃/s, etc. The cooling rate may be at least 0.1℃/s. For example, the cooling rate may be at least 0.5℃/s, 1℃/s, 2℃/s, 3℃/s, 4℃/s, 5℃/s, 6℃/s, 7℃/s, 8℃/s, 9℃/s, 10℃/s, 11℃/s, 12℃/s, 13℃/s, 14℃/s, 15℃/s, 16℃/s, 17℃/s, 18℃/s, 19℃/s, 20℃/s, 21℃/s, etc.

In some embodiments, the controller does not subject the reaction mixture to the one or more cycles of heating and cooling in the absence of the switch being turned on. For example, the controller may subject the reaction mixture to heating or cooling only upon the switch being turned on. In some embodiments, the controller subjects the reaction mixture to one or more cycles of heating and cooling after a time delay upon the switch being turned on.

In some embodiments, prior to activating the system, the method may further comprise securing the reaction vessel to a sample holder mounted to the heating member. The reaction chamber of a reaction vessel may be adjacent to and in thermal communication with the heating member when the reaction vessel is secured to the sample holder. In some embodiments, the sample holder is mounted to the heating member； this may ensure that the reaction vessel or the sample therein is in thermal communication with the heating member when secured in or on the sample holder.

A method of the present disclosure may further comprise providing excitation energy to the reaction vessel to induce a signal (s) that is indicative of a presence or absence of an amplification product of the nucleic acid amplification reaction. The excitation energy may be provided from a light source, or other inductive energy sources, e.g., a light-emitting diode, a laser or other energy sources. The excitation energy may be directed to the sample comprised in the reaction chamber, thereby generating emitted signals (e.g., optical signals, fluorescent signals and/or electrostatic signals) indicating occurrence and/or result of the chemical or biological reaction (e.g., nucleic acid amplification reaction) on the sample.

In a method of the present disclosure, one or more sensors (e.g., 2, 3, 4, 5, 6, 7 or more sensors) may be provided. For example, the method may comprise providing a first sensor in sensing communication with the reaction vessel to detect the signal (s) that is indicative of the presence or absence of the amplification product. The first sensor may be an optical sensor, an inductive sensor, an electrochemical sensor, an electrostatic sensor, and/or an impedance sensor. The signal (s) may be an optical signal, a fluorescent signal and/or an electrostatic signal.

In some embodiments, the method comprises providing a second sensor in sensing communication with the reaction vessel to detect a temperature at one or more location (s) within the reaction chamber of the reaction vessel. The second sensor may detect a temperature at one or more location (s) within the reaction chamber of the reaction vessel.

The method may comprise providing one or more (e.g., 1, 2, 3, or more) display (s) operatively coupled to the first sensor and/or the second sensor in the base and/or the housing. The display may be configured to display the signal (s) indicative of the presence or absence of the amplification product, and/or the temperature at the one or more location (s) within the reaction chamber of the reaction vessel.

Prior to activating the system, the base having the housing mounted thereto may be deposited in a refrigeration unit (e.g., a refrigerator, a cold room, etc. ) .

In a method of the present disclosure, thermal energy may be provided to the reaction chamber until the reaction mixture reaches a first temperature, and the convective fluid may be subjected to flow across the reaction chamber until the reaction mixture reaches a second temperature that is less than the first temperature. The method may further comprise reducing or terminating flow of the convective fluid when the reaction mixture reaches the second temperature.

The reaction vessel may comprise a sampling unit； the sampling unit may comprise a collection member and a sampling chamber in fluid communication with the reaction chamber. The nucleic acid sample may be deposited in the reaction vessel by piercing a sealing member sealing an opening of the sampling chamber with the collection member having collected the nucleic acid sample thereon or therein.

In another aspect, the present disclosure provides a system for nucleic acid amplification. The system may comprise a base comprising a heating member, and the base may have a footprint that is less than or equal to about 9000 mm². For example, the base may have a footprint that is less than or equal to about 8000 mm², about 7000 mm², about 6000 mm², about 5000 mm², about 4500 mm², about 4000 mm², about 3500 mm², about 3000 mm², about 2500 mm², about 2000 mm², about 1500 mm², about 1000 mm², about 900 mm², about 800 mm², about 700 mm², about 600 mm², about 500 mm², about 400 mm², about 300 mm², about 200 mm², about 100 mm², etc.

The system may comprise a reaction vessel comprising a reaction chamber. The reaction chamber may be adjacent to and in thermal communication with the heating member. During use, the heating member may provide thermal energy to a reaction mixture in the reaction chamber； the reaction mixture may comprise a nucleic acid sample and reagents necessary for nucleic acid amplification. The reaction chamber may have a footprint that is less than or equal to about 2000 mm². For example, the reaction chamber may have a footprint that is less than or equal to about 1500 mm², about 1000 mm², about 900 mm², about 800 mm², about 700 mm², about 600 mm², about 500 mm², about 400 mm², about 300 mm², about 200 mm², about 100 mm², etc. In some embodiments, a cross-section of the reaction chamber is less than a cross-section of the heating member. In some embodiments, a footprint of the reaction chamber is less than a footprint of the heating member.

The system may comprise a housing removably mountable to the base. The housing may at least partially or completely encapsulate the reaction vessel when mounted to the base, and the housing may include a cooling member that is in thermal communication with the reaction vessel when the housing is mounted to the base.

The system may comprise a controller operatively coupled to the heating member. The controller may be programmed to subject the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture. This may be performed by regulating (i) a rate of heating of the reaction vessel using the heating member and (ii) a rate of cooling of the reaction vessel using the cooling member.

In another aspect, the present disclosure provides a method for nucleic acid amplification. The method may comprise activating a system comprising (i) a base comprising a heating member, the base may have a footprint that is less than or equal to about 5000 mm², (ii) a reaction vessel comprising a reaction chamber, the reaction chamber may be adjacent to and in thermal communication with the heating member, and the reaction chamber may comprise a reaction mixture comprising a nucleic acid sample and reagents necessary for nucleic acid amplification, and (iii) a housing removably mounted to the base, the housing may encapsulate the reaction vessel, and the housing may include a cooling member that is in thermal communication with the reaction vessel when the housing is mounted to the base. The method may further comprise subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture, this may be achieved by regulating (i) a rate of heating of the reaction vessel using the heating member and (ii) a rate of cooling of the reaction vessel using the cooling member. In some embodiments, the base has a footprint that is less than or equal to about 9000 mm², 8000 mm², about 7000 mm², about 6000 mm², about 5000 mm², about 4500 mm², about 4000 mm², about 3500 mm², about 3000 mm², about 2500 mm², about 2000 mm², about 1500 mm², about 1000 mm², about 900 mm², about 800 mm², about 700 mm², about 600 mm², about 500 mm², about 400 mm², about 300 mm², about 200 mm², about 100 mm², etc.

The cooling member may be formed of a material with a heat capacity of at least about 0.1 J /g*K, e.g., at least about 0.2 J /g*K, at least about 0.3 J /g*K, at least about 0.4 J /g*K, at least about 0.5 J /g*K, at least about 1.0 J /g*K, at least about 1.5J /g*K, at least about 2.0 J /g*K, at least about 2.5 J /g*K or more. In some embodiments, the cooling member is a solid comprising copper.

The nucleic acid sample may be a biological sample, such as a biological sample obtained from a subject. There are various approaches for obtaining a biological sample from a subject. Non-limiting examples of obtaining a biological sample directly from a subject include accessing the circulatory system (e.g., intravenously or intra-arterially via a syringe or other needle) , collecting a secreted biological sample (e.g., feces, urine, sputum, saliva, etc. ) , surgically (e.g., biopsy) , swabbing (e.g., buccal swab, oropharyngeal swab) , pipetting, and breathing. Moreover, a biological sample may be obtained from any anatomical part of a subject where a desired biological sample is located.

A biological sample obtained directly from a subject may generally refer to a biological sample that has not been further processed after being obtained from the subject, with the exception of collecting the biological sample from the subject for further processing. For example, blood may be obtained directly from a subject by accessing the subject’s circulatory system, removing the blood from the subject (e.g., via a needle) , and entering the removed blood into a receptacle. The receptacle may comprise reagents (e.g., anti-coagulants) such that the blood sample is useful for further analysis. In another example, a swab may be used to access epithelial cells on an oropharyngeal surface of the subject. After obtaining the biological sample from the subject, the swab containing the biological sample can be contacted with a fluid (e.g., a buffer) to collect the biological fluid from the swab. Alternatively, pre-processing may occur on the biological sample prior to being provided to the system.

In some embodiments, a biological sample has not been purified when provided in a reaction vessel. In some embodiments, the nucleic acid of a biological sample has not been extracted when the biological sample is provided to a reaction vessel. For example, the RNA or DNA in a biological sample may not be extracted from the biological sample when providing the biological sample to a reaction vessel. Moreover, in some embodiments, a target nucleic acid (e.g., a target RNA or target DNA) present in a biological sample may not be concentrated prior to providing the biological sample to a reaction vessel. Alternatively, dilution or concentration of the sample may occur prior to being provided to a system.

The reaction mixture may also include an agent that detects amplified target nucleic acid. The agent may be a reporter agent that can yield a detectable signal whose presence or absence is indicative of the presence of an amplified product. The intensity of the detectable signal may be proportional to the amount of amplified product. For example, the detectable signal may be directly linearly proportional, exponentially proportional, reversely proportional, or have any other type of proportional relationship to the amount of amplified product. In some cases, where amplified product is generated of a different type of nucleic acid than the target nucleic acid initially amplified, the intensity of the detectable signal is proportional to the amount of target nucleic acid initially amplified. For example, in the case of amplifying a target RNA via parallel reverse transcription and amplification of the DNA obtained from reverse transcription, reagents necessary for both reactions may also comprise a reporter agent may yield a detectable signal that is indicative of the presence of the amplified DNA product and/or the target RNA amplified. The intensity of the detectable signal may be proportional to the amount of the amplified DNA product and/or the original target RNA amplified. The use of a reporter agent also enables real-time amplification methods, including real-time PCR for DNA amplification.

Reporter agents may be linked with nucleic acids, including amplified products, by covalent or non-covalent bonds. Non-limiting examples of non-covalent bonds include ionic interactions, Van der Waals forces, hydrophobic interactions, hydrogen bonding, and combinations thereof. In some embodiments, reporter agents bind to initial reactants and changes in reporter agent levels may be used to detect amplified product. In some embodiments, reporter agents is only detectable (or non-detectable) as nucleic acid amplification progresses. In some embodiments, an optically-active dye (e.g., a fluorescent dye) is used as may be used as a reporter agent. An agent for detecting amplified target nucleic acid may be a nucleic acid binding dye. The dye may be a DNA-intercalating dye. Non-limiting examples of dyes include Eva green, SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, phenanthridines and acridines, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue) , SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green) , SYTO-81, -80, -82, -83, -84, -85 (orange) , SYTO-64, -17, -59, -61, -62, -60, -63 (red) , fluorescein, fluorescein isothiocyanate (FITC) , tetramethyl rhodamine isothiocyanate (TRITC) , rhodamine, tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, , Cy-7, Texas Red, Phar-Red, allophycocyanin (APC) , Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, cascade blue, dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes such as those including europium and terbium, carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein (FAM) , 5- (or 6-) iodoacetamidofluorescein, 5- { [2 (and 3) -5- (Acetylmercapto) -succinyl] amino} fluorescein (SAMSA-fluorescein) , lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine (ROX) , 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid (AMCA) , BODIPY fluorophores, 8-methoxypyrene-1, 3, 6-trisulfonic acid trisodium salt, 3, 6-Disulfonate-4-amino-naphthalimide, phycobiliproteins, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or other fluorophores.

In some embodiments, a reporter agent is a sequence-specific oligonucleotide probe that can be optically active when hybridized with an amplified product. Due to sequence-specific binding of the probe to the amplified product, use of oligonucleotide probes can increase specificity and sensitivity of detection. A probe may be linked to any of the optically-active reporter agents (e.g., dyes) described herein and may also include a quencher capable of blocking the optical activity of an associated dye. Non-limiting examples of probes that may be useful used as reporter agents include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

A reporter agent may be an RNA oligonucleotide probe that may include an optically-active dye (e.g., fluorescent dye) and a quencher positioned adjacently on the probe. The close proximity of the dye with the quencher can block the optical activity of the dye. The probe may bind to a target sequence to be amplified. Upon the breakdown of the probe with the exonuclease activity of a DNA polymerase during amplification, the quencher and dye are separated, and the free dye regains its optical activity that can subsequently be detected.

A reporter agent may be a molecular beacon. A molecular beacon may include, for example, a quencher linked at one end of an oligonucleotide in a hairpin conformation. At the other end of the oligonucleotide is an optically active dye, such as, for example, a fluorescent dye. In the hairpin configuration, the optically-active dye and quencher are brought in close enough proximity such that the quencher is capable of blocking the optical activity of the dye. Upon hybridizing with amplified product, however, the oligonucleotide assumes a linear conformation and hybridizes with a target sequence on the amplified product. Linearization of the oligonucleotide results in separation of the optically-active dye and quencher, such that the optical activity is restored and can be detected. The sequence specificity of the molecular beacon for a target sequence on the amplified product can improve specificity and sensitivity of detection.

In some embodiments, a reporter agent is a radioactive species. Non-limiting examples of radioactive species include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, Tc99m, ³⁵S, or ³H.

In some embodiments, a reporter agent is an enzyme that is capable of generating a detectable signal. Detectable signal may be produced by activity of the enzyme with its substrate or a particular substrate in the case the enzyme has multiple substrates. Non-limiting examples of enzymes that may be used as reporter agents include alkaline phosphatase, horseradish peroxidase, I²-galactosidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, and luciferase.

The nucleic acid sample may be provided with reagents necessary for nucleic acid amplification within the reaction vessel. In some embodiments, a reagent comprises one or more of the following: (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for the target nucleic acid (e.g., RNA) . Some examples of reagents may include a commercially available pre-mixture (e.g., Qiagen One-Step RT-PCR or One-Step RT-qPCR kit) comprising reverse transcriptases (e.g., Sensiscript and Omniscript transcriptases) , a DNA Polymerase (e.g., HotStarTaq DNA Polymerase) , and dNTPs.

In some embodiments, the sample is provided within a sample container, such as a reaction vessel. Any components of the sample including the target nucleic acid, agent that detects amplified target nucleic acid, and/or reagents for nucleic acid amplification may be provided within the reaction vessel to obtain a reaction mixture. Any suitable reaction vessel may be used. In some embodiments, a reaction vessel comprises a body that can include an interior surface, an exterior surface, an open end, and an opposing closed end. In some embodiments, a reaction vessel comprises a cap. The cap may be configured to contact the body at its open end, such that when contact is made the open end of the reaction vessel is closed. In some cases, the cap is permanently associated with the reaction vessel such that it remains attached to the reaction vessel in open and closed configurations. In some cases, the cap is removable, such that when the reaction vessel is open, the cap is separated from the reaction vessel. In some embodiments, a reaction vessel may be sealed, optionally hermetically sealed. The reaction vessel may be fluid-tight.

A reaction vessel may comprise a body and a cap； the cap may be removably attached to the body or permanently associated with the body. The body may comprise one or more walls forming a reaction chamber and a sampling chamber, the sampling chamber may be in fluid communication with the reaction chamber. In some embodiments, the reaction chamber and the sampling chamber are at least partially separated from each other spatially. For example, there might be a seal between the reaction chamber and the sampling chamber that at least partially prevents a fluid from flowing between the reaction chamber and the sampling chamber. In some embodiments, the seal between the reaction chamber and the sampling chamber may be penetrated (e.g. pierced) to form an opening, so that a fluid may flow from the reaction chamber to the sampling chamber or vice versa. The sampling chamber may comprise an opening, and a sealing member may be positioned at the opening or inside the sampling chamber separating contents (e.g., reaction mixture) within the chamber from the environment outside the chamber. The cap may comprise a collection member (e.g., a needle or a notch) configured to access and retain a sample. The dimensions of the collection member and the opening of the sampling chamber are configured in a way that when the opening is closed with the cap, the collection member may fit into the opening, pierce through the sealing member and release the sample retained thereon or therein into the sampling chamber (e.g., into the reaction mixtures or other solutions contained in the sampling chamber) .

Any dimensions may be provided for a reaction vessel. The reaction vessel may be configured to have a volume to contain no more than about 100 μL of a reaction mixture. For example, the reaction vessel may be configured to have a volume to contain no more than about 0.01 μL, 0.03 μL, 0.05 μL, 0.07 μL, 0.1 μL, 0.5 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, or 200 μL reaction mixture. The reaction vessel may have a volume configured to contain no more than a volume falling into a range between any two of the values described herein.

The reaction vessel may have a height that is less than or equal to about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 50 mm, 60 mm, or 70 mm. The reaction vessel may have a height falling into a range between any two of the values described herein.

The reaction vessel may have a cross-sectional area of at least about 1 mm², 5 mm², 10 mm², 20 mm², 30 mm², 40 mm², 50 mm², 60 mm², 70 mm², 80 mm², 90 mm², 100 mm², 150 mm², 200 mm², 250 mm², 300 mm², 350 mm², 400 mm², 450 mm², 500 mm², 550 mm², 600 mm², 650 mm², 700 mm², 750 mm², 800 mm², 850 mm², 900 mm², 950 mm², 1000 mm², 1100 mm², 1200 mm², 1300 mm², 1400 mm², or 1500 mm². The reaction vessel may have a cross-sectional area falling into a range between any two of the values described herein.

Walls of a reaction vessel may have a thickness that is no more than about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.15 mm, about 0.2 mm, about 0.25 mm, about 0.3 mm, about 0.35 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1.0 mm, about 2.0 mm, about 3.0 mm, about 4.0 mm, about 5.0 mm, about 6.0 mm, about 7.0 mm, about 8.0 mm, about 9.0 mm, about 10 mm, about 15 mm, about 20 mm, etc.

Reaction vessels may be constructed of any suitable material with non-limiting examples of such materials that include glasses, metals, plastics, and combinations thereof. Reaction vessels can be made from optically transparent or translucent materials that may permit an optical signal from within the reaction vessel to leave the reaction vessel. The reaction vessels may be made from a material that may or may not filter an optical signal exiting the reaction vessel. In some embodiments, the reaction vessels are formed from a clear material that may permit a detector to view the interior of the reaction vessels. In some embodiments, the interior of the reaction vessels may be imaged. Alternatively, an amount of optical signal exiting the reaction vessel may be detected and measured.

A sample holder may be capable of receiving a reaction vessel. The reaction vessels may be removably provided to the sample holder. The reaction vessels may be inserted within a sample holder or taken out of the sample holder. The reaction vessels may be placed onto a supporting component of the system of the present disclosure or taken off from the supporting component.

Time may elapse while nucleic acid amplification reactions are occurring. The system may comprise a detector capable of detecting a signal during the time while the nucleic acid amplification reaction is occurring. The detector may be capable of detecting the signal without removing the sample from the system.

In any of the various aspects, the detector may detect amplified product (e.g., amplified DNA product, amplified RNA product) . Detection of amplified product, including amplified DNA, may be accomplished with any suitable detection method known in the art. The particular type of detection method used may depend, for example, on the particular amplified product, the type of reaction vessel used for amplification, other reagents in a reaction mixture, whether or not a reporter agent was included in a reaction mixture, and if a reporter agent was used, the particular type of reporter agent used. Non-limiting examples of detection methods include optical detection, spectroscopic detection, electrostatic detection, electrochemical detection, etc. Optical detection methods include, but are not limited to, fluorimetry and UV-vis light absorbance. Spectroscopic detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel based techniques, such as, for example, gel electrophoresis. Electrochemical detection methods include, but are not limited to, electrochemical detection of amplified product after high-performance liquid chromatography separation of the amplified products.

The detector may be capable of detecting an optical signal from the sample. The optical signal may be a fluorescent or other luminescent signal from the sample. The optical signal may be generated by the sample in response to a stimulation light provided to the sample. A stimulation light may be provided by a light source. The light source may be within the system. In some embodiments, light is absorbed by the sample, and the sample emits light. The emitted light may be at the same or different wavelength from the absorbed light. In some embodiments, the optical signal is a reflection of light from the light source. Alternatively, light may be shined through the sample, and the detector may be capable of detecting the light that passes through the sample.

In some embodiments, information regarding the presence of and/or an amount of amplified product (s) (e.g., amplified DNA product) is outputted to a recipient. There are various ways to output information regarding amplified product (s) . Such information may be provided in real-time while the nucleic-acid amplification is underway. In other instances, the information may be provided once the nucleic acid amplification has been completed. In some embodiments, some data may be provided in real-time while other data may be presented once the amplification is completed.

In some embodiments, such information is provided visually (e.g., on a display) or verbally (e.g., by a medical practitioner) to a recipient. In some embodiments, such information is provided in a report. A report may include any number of desired elements, with non-limiting examples that include information regarding the subject (e.g., sex, age, race, health status, etc. ) raw data, processed data (e.g. graphical displays (e.g., figures, charts, data tables, data summaries) , determined cycle threshold values, calculation of starting amount of target polynucleotide) , conclusions about a presence of the target nucleic acid, diagnosis information, prognosis information, disease information, etc., and combinations thereof. The report may be provided as a printed report (e.g., a hard copy) or may be provided as an electronic report. In some embodiments, including cases where an electronic report is provided, such information is outputted via an electronic display, such as a monitor or television, a screen operatively linked with a unit used to obtain the amplified product, a tablet computer screen, a mobile system screen, etc. Both printed and electronic reports may be stored in files or in databases, respectively, such that they are accessible for comparison with future reports.

A report may be transmitted to the recipient at a local or remote location using any suitable communication medium including, for example, a network connection, a wireless connection, the cloud, or an internet connection. In some embodiments, a report is sent to a recipient’s system, such as a personal computer, phone, tablet, or other system. The report may be viewed online, saved on the recipient’s system, or printed. There are other suitable approaches for transmitting a report, with non-limiting examples that include mailing a hard-copy report for reception and/or for review by a recipient.

The report or information contained in a report may be outputted to various types of recipients. Non-limiting examples of such recipients include the subject from which the biological sample was obtained, a physician, a physician treating the subject, a clinical monitor for a clinical trial, a nurse, a researcher, a laboratory technician, a representative of a pharmaceutical company, a health care company, a biotechnology company, a hospital, a human aid organization, a health care manager, an electronic system (e.g., one or more computers and/or one or more computer servers storing, for example, a subject’s medical records) , a public health worker, other medical personnel, and other medical facilities.

The housing may partially or completely enclose components of the system (e.g., the reaction vessel) . The housing may surround components of the system (e.g., the reaction vessel) laterally and/or on the top and bottom. The housing may be a flexible or a rigid structure. The detector may be contained within the housing. In some embodiments, the detector is located outside the housing of the system. The detector may be an integral part of the system. Alternatively, the detector may be removable or separable from the system.

An optical path may be provided between the sample and the detector. A signal from the sample may reach the detector via the optical path. An optical signal from a sample may traverse the optical path to reach the detector. The optical path may include direct line-of-sight between the sample and the detector. In some embodiments, one or more optical elements may be provided between the sample and the detector. Examples of optical elements may include lenses, mirrors, prisms, diffusers, concentrators, filters, dichroics, optical fibers, or any other type of optical elements.

The optical path may be provided entirely within a housing of the system. The housing may optically isolate the optical path from the surrounding environment. For example, the housing may be light-tight so that little or no interfering optical signals may be provided within the housing that may interfere with the optical path. Light from outside the housing may not be capable of entering the interior of the housing. This may advantageously reduce inaccuracies in the optical signal detected by the detector.

The optical path may remain while the nucleic acid amplification is occurring. The detector may be able to continuously or periodically detect signals from the sample while the nucleic acid amplification is occurring via the optical path.

In some embodiments, a battery pack is used as a power supply to power the system. The battery pack may be used to power the heating member, the fluid flow member, and/or the detector.

The power supply may provide a low voltage for the system of the present disclosure. In some embodiment, the low voltage is less than or equal to about 60 V, 50 V, 48 V, 40 V, 30 V, 24 V, 20 V, 18 V, 16 V, 15 V, 14 V, 13 V, 12V, 11 V, 10V, 9 V, 8V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, or 1 V. In some embodiments, a low voltage of less than or equal to about 50 V, 40 V, 30 V, 24 V, 20 V, 18 V, 16 V, 15 V, 14 V, 13 V, 12V, 11 V, 10V, 9 V, 8V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, or 1 V is used to supply power to the heating member, the fluid flow member, the excitation energy source, and/or the detector.

In some embodiments, a low degree of power may be used for operating the system or conducting the method of the present disclosure. For example, about 84 W may be used to operate the system or conduct the method of the present disclosure. In some embodiments, a low power is less than or equal to about 250 W, 200 W, 150 W, 130 W, 120 W, 110 W, 100 W, 90 W, 85 W, 84 W, 83 W, 80 W, 75 W, 70 W, 65 W, 60 W, 55 W, 50 W, 45 W, 40 W, 35 W, 30 W, 25 W, 20 W, 15 W, 10 W, 5 W, 1 W, 500 mW, 100 mW, 50 mW, 10 mW, 5 mW, or 1 mW. The amount of power used to operate the system or conduct the method may fall into a range between any two of the values described herein.

In various aspects, primer extension reactions are utilized to generate amplified product. Primer extension reactions generally comprise a cycle of incubating a reaction mixture at a first temperature for a first duration and incubating a reaction mixture at a second temperature that is less than the first temperature for a second duration. The first temperature may be a denaturation temperature. The first duration may be a denaturation duration. The second temperature may be an elongation temperature. The second duration may be an elongation duration.

Denaturation temperatures may vary depending upon, for example, the particular nucleic acid sample analyzed, the particular source of target nucleic acid (e.g., viral particle, bacteria) in the nucleic acid sample, the reagents used, and/or the desired reaction conditions. For example, a denaturation temperature may be from about 80℃ to about 110℃. In some examples, a denaturation temperature may be from about 90℃ to about 100℃. In some examples, a denaturation temperature may be from about 90℃ to about 97℃. In some examples, a denaturation temperature may be from about 92℃ to about 95℃. In still other examples, a denaturation temperature may be at least or equal to about 80°, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, or 100℃.

Denaturation durations may vary depending upon, for example, the particular nucleic acid sample analyzed, the particular source of target nucleic acid (e.g., viral particle, bacteria) in the nucleic acid sample, the reagents used, and/or the desired reaction conditions. For example, a denaturation duration may be less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, a denaturation duration may be no more than 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

Elongation temperatures may vary depending upon, for example, the particular nucleic acid sample analyzed, the particular source of target nucleic acid (e.g., viral particle, bacteria) in the nucleic acid sample, the reagents used, and/or the desired reaction conditions. For example, an elongation temperature may be from about 30℃ to about 80℃. In some examples, an elongation temperature may be from about 35℃ to about 72℃. In some examples, an elongation temperature may be from about 45℃ to about 65℃. In some examples, an elongation temperature may be from about 35℃ to about 65℃. In some examples, an elongation temperature may be from about 40℃ to about 60℃. In some examples, an elongation temperature may be from about 50℃ to about 60℃. In still other examples, an elongation temperature may be no more than or equal to about 35°, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, or 80℃.

Elongation durations may vary depending upon, for example, the particular nucleic acid sample analyzed, the particular source of target nucleic acid (e.g., viral particle, bacteria) in the nucleic acid sample, the reagents used, and/or the desired reaction conditions. For example, an elongation duration may be less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, an elongation duration may be no more than 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

In any of the various aspects, multiple cycles of a primer extension reaction can be conducted. Any suitable number of cycles may be conducted. For example, the number of cycles conducted may be less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 cycles. The number of cycles conducted may depend upon, for example, the number of cycles (e.g., cycle threshold value (Ct) ) necessary to obtain a detectable amplified product (e.g., a detectable amount of amplified DNA product that is indicative of the presence of a target RNA in a nucleic acid sample) . For example, the number of cycles necessary to obtain a detectable amplified product (e.g., a detectable amount of DNA product that is indicative of the presence of a target RNA in a nucleic acid sample) may be less than or equal to about 100 cycles, 75 cycles, 70 cycles, 65 cycles, 60 cycles, 55 cycles, 50 cycles, 40 cycles, 35 cycles, 30 cycles, 25 cycles, 20 cycles, 15 cycles, 10 cycles, or 5 cycles. Moreover, in some embodiments, a detectable amount of an amplifiable product (e.g., a detectable amount of DNA product that is indicative of the presence of a target RNA in a biological sample) is obtained at a cycle threshold value (Ct) of less than about 100, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5.

The time for which amplification yields a detectable amount of amplified product indicative of the presence of a target nucleic acid amplified can vary depending upon the nucleic acid sample from which the target nucleic acid was obtained, the particular nucleic acid amplification reactions to be conducted, and the particular number of cycles of amplification reaction desired. For example, amplification of a target nucleic acid may yield a detectable amount of amplified product indicative to the presence of the target nucleic acid at time period of 120 minutes or less； 90 minutes or less； 60 minutes or less； 50 minutes or less； 45 minutes or less； 40 minutes or less； 35 minutes or less； 30 minutes or less； 25 minutes or less； 20 minutes or less； 15 minutes or less； 10 minutes or less； or 5 minutes or less.

In some embodiments, amplification of a target RNA yields a detectable amount of amplified DNA product indicative of a presence of the target RNA at time period of 120 minutes or less； 90 minutes or less； 60 minutes or less； 50 minutes or less； 45 minutes or less； 40 minutes or less； 35 minutes or less； 30 minutes or less； 25 minutes or less； 20 minutes or less； 15 minutes or less； 10 minutes or less； or 5 minutes or less.

In some embodiments, a reaction mixture is subjected to a plurality of series of primer extension reactions. An individual series of the plurality may comprise multiple cycles of a particular primer extension reaction, characterized, for example, by particular denaturation and elongation conditions as described elsewhere herein. Generally, each individual series differs from at least one other individual series in the plurality with respect to, for example, a denaturation condition and/or elongation condition. An individual series may differ from another individual series in a plurality of series, for example, with respect to any one, two, three, or all four of denaturing temperature, denaturing duration, elongation temperature, and elongation duration. Moreover, a plurality of series may comprise any number of individual series such as, for example, at least about or about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more individual series.

For example, a plurality of series of primer extension reactions may comprise a first series and a second series. The first series, for example, may comprise more than ten cycles of a primer extension reaction, where each cycle of the first series comprises (i) incubating a reaction mixture at about 92℃ to about 95℃ for no more than 30 seconds followed by (ii) incubating the reaction mixture at about 35℃ to about 65℃ for no more than about one minute. The second series, for example, may comprise more than ten cycles of a primer extension reaction, where each cycle of the second series comprises (i) incubating the reaction mixture at about 92℃ to about 95℃ for no more than 30 seconds followed by (ii) incubating the reaction mixture at about 40℃ to about 60℃ for no more than about 1 minute. In this particular example, the first and second series differ in their elongation temperature condition. The example, however, is not meant to be limiting as any combination of different elongation and denaturing conditions could be used.

In some embodiments, the ramping time (i.e., the time the system takes to transition from one temperature to another) and/or ramping rate can be important factors in amplification. For example, the temperature and time for which amplification yields a detectable amount of amplified product indicative of the presence of a target nucleic acid can vary depending upon the ramping rate and/or ramping time. The ramping rate can impact the temperature (s) and time (s) used for amplification.

The ramping time and/or ramping rate may be different between cycles. In some embodiments, however, the ramping time and/or ramping rate between cycles are the same. The ramping time and/or ramping rate can be adjusted based on the sample (s) that are being processed.

In some embodiments, the ramping time between different temperatures are determined, for example, based on the nature of the sample and the reaction conditions. The exact temperature and incubation time can also be determined based on the nature of the sample and the reaction conditions. In some embodiments, a single sample is processed (e.g., subjected to amplification conditions) multiple times using multiple thermal cycles, with each thermal cycle differing for example by the ramping time, temperature, and/or incubation time. The best or optimum thermal cycle can then be chosen for that particular sample. This provides a robust and efficient method of tailoring the thermal cycles to the specific sample or combination of samples being tested.

In some embodiments, a target nucleic acid is subjected to a denaturing condition prior to initiation of a primer extension reaction. In the case of a plurality of series of primer extension reactions, the target nucleic acid may be subjected to a denaturing condition prior to executing the plurality of series or may be subjected to a denaturing condition between series of the plurality. For example, the target nucleic acid may be subjected to a denaturing condition between a first series and a second series of a plurality of series. Non-limiting examples of such denaturing conditions include a denaturing temperature profile (e.g., one or more denaturing temperatures) and a denaturing agent.

In some embodiments, a nucleic acid sample is preheated prior to conducting a primer extension reaction. The temperature (e.g., a preheating temperature) at which and duration (e.g., a preheating duration) for which a nucleic acid sample is preheated may vary depending upon, for example, the particular nucleic acid sample being analyzed. In some examples, a nucleic acid sample is preheated for no more than about 60 minutes, 50 minutes, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or 5 seconds. In some examples, a nucleic acid sample is preheated at a temperature from about 80℃ to about 110℃. In some examples, a nucleic acid sample is preheated at a temperature from about 90℃ to about 100℃. In some examples, a nucleic acid sample is preheated at a temperature from about 90℃ to about 97℃. In some examples, a nucleic acid sample is preheated at a temperature from about 92℃ to about 95℃. In some examples, a nucleic acid sample is preheated at a temperature of no more than or equal to about 80°, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, or 100℃.

In any of the various aspects, the time required to complete the elements of a method may vary depending upon the particular steps of the method. For example, an amount of time for completing the elements of a method may be from about 5 minutes to about 120 minutes. In other examples, an amount of time for completing the elements of a method may be from about 5 minutes to about 60 minutes. In other examples, an amount of time for completing the elements of a method may be from about 5 minutes to about 30 minutes. In other examples, an amount of time for completing the elements of a method may be less than or equal to 120 minutes, less than or equal to 90 minutes, less than or equal to 75 minutes, less than or equal to 60 minutes, less than or equal to 45 minutes, less than or equal to 40 minutes, less than or equal to 35 minutes, less than or equal to 30 minutes, less than or equal to 25 minutes, less than or equal to 20 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes, or less than or equal to 5 minutes.

The system is capable of controlling a temperature of a sample precisely to achieve a desired temperature profile. The system may be capable of controlling the temperature to within about plus or minus 5 ℃, 4 ℃, 3 ℃, 2 ℃, 1.2 ℃, 1 ℃, 0.7 ℃, 0.5 ℃, 0.3 ℃, 0.1 ℃, 0.05 ℃, 0.01 ℃, 0.005 ℃, or 0.001 ℃. The system may advantageously be capable of providing high quality temperature control while operating at a low voltage and/or low power. The system may be capable of delivering high quality temperature control while having small dimensions.

Detection of signals from the sample undergoing amplification may occur throughout the process. The detection may occur continuously or at one or more points during the amplification process. The sample may emit optical signals throughout the process. The optical signals may be related to the amount of amplified target nucleic acid in the sample.

Data relating to the detected signals may be displayed in real-time. For example, data relating to the progress of the nucleic acid amplification and/or results of the nucleic acid amplification may be displayed while amplification is occurring. In some embodiments, one or more display (s) may be built-into the system. For example, the display may be provided on/in a housing and/or a base of the system. Any description of a display may apply to any type of output module. The display may include a visual display, as well as audio or tactile output of information. The display may show information on a screen or other type of user interface (UI) . For example, a screen may be built into the system.

In some embodiments, the data is shown on a separate display device. The separate display device may communicate with the system. In some embodiments, communications may occur via a connection. The connection may be a hard-wired connection or a wireless connection. Direct communications may occur between the system and the display device. For example, Bluetooth, infra-red communications, radio, WiFi, or other direct communications may occur. In other instances, indirect communications may occur between the system and the display device. For examples, communications may occur over a network, such as a local area network (LAN) , or wide area network (WAN) such as the Internet. In some embodiments, telecommunications networks are used (e.g., cellular phone networks, data networks) . In some examples, 3G, 4G or 5G networks are used for communications. One or more intermediate systems, such as relay systems (e.g., towers) or routers, may be used in communications. Alternatively, no intermediate system is used.

A system may have an input module that receives a user request to amplify a target nucleic acid (e.g., target RNA, target DNA) present in a nucleic acid sample obtained from a subject. Any suitable module capable of accepting such a user request may be used. The input module may comprise, for example, a device that comprises one or more processors. The input module may be built into the system. The input module may be integrated into a housing or base of the system and/or accessible from outside the housing.

Alternatively, the input module may be separate or separable from the system. The input module may communicate with the system over a connection, such as those described elsewhere in the present disclosure. Non-limiting examples of systems that comprise processors include a desktop computer, a laptop computer, a tablet computer (e.g.,

iPad,

Galaxy Tab) , a cell phone, a smart phone (e.g.,

iPhone,

enabled phone) , a personal digital assistant (PDA) , a video-game console, a television, a music playback system (e.g.,

iPod) , a video playback system, a pager, and a calculator. Processors may be associated with one or more controllers, calculation units, and/or other units of a computer system, or implanted in firmware as desired. If implemented in software, the routines (or programs) may be stored in any computer readable memory such as in RAM, ROM, flash memory, a magnetic disk, a laser disk, or other storage medium. Likewise, this software may be delivered to a system via any known delivery method including, for example, over a communication channel such as a telephone line, the internet, a local intranet, a wireless connection, etc., or via a transportable medium, such as a computer readable disk, flash drive, etc. The various steps may be implemented as various blocks, operations, tools, modules or techniques which, in turn, may be implemented in hardware, firmware, software, or any combination thereof. When implemented in hardware, some or all of the blocks, operations, techniques, etc. may be implemented in, for example, a custom integrated circuit (IC) , an application specific integrated circuit (ASIC) , a field programmable logic array (FPGA) , a programmable logic array (PLA) , etc.

In some embodiments, the input module is configured to receive a user request to perform amplification of the target nucleic acid in an amplification module. The amplification module may comprise components for completing one or more cycles of heating and cooling, and/or one or more primer extension reaction. The input module may receive the user request directly (e.g. by way of an input system such as a keyboard, mouse, or touch screen operated by the user) or indirectly (e.g. through a wired or wireless connection, including over the internet) . Via output electronics, the input module may provide the user’s request to the amplification module. In some embodiments, an input module may include a user interface (UI) , such as a graphical user interface (GUI) , which is configured to enable a user to provide a request to amplify the target nucleic acid. A GUI can include textual, graphical and/or audio components. A GUI can be provided on an electronic display, including the display of a system comprising a computer processor. Such a display may include a resistive or capacitive touch screen.

In some embodiments, the output module comprises a system with a processor as described for the input module. The output module may include input devices as described herein and/or may comprise input electronics for communication with the amplification module. In some embodiments, the output module is an electronic display, such as a display on a nucleic acid amplification system or a separate display device. In some embodiments, the electronic display comprises a UI. In some embodiments, the output module is a communication interface operatively coupled to a computer network, such as the internet. In some embodiments, the output module transmits information to a recipient at a local or remote location using any suitable communication medium, including a computer network, a wireless network, the cloud, a local intranet, or the internet. In some embodiments, the output module is capable of analyzing data received from the amplification module. The output module may analyze information in real-time while amplification is occurring. Some data may be analyzed after the amplification has been completed. In some cases, the output module includes a report generator capable of generating a report and transmitting the report to a recipient, the report may contain any information regarding the amount and/or presence of amplified product as described elsewhere herein. In some embodiments, the output module may transmit information automatically in response to information received from the amplification module, such as in the form of raw data or data analysis performed by software included in the amplification module. Alternatively, the output module may transmit information after receiving instructions from a user. Information transmitted by the output module may be viewed electronically or printed from a printer.

One or more of the input module, amplification module, and output module may be contained within the same system or may comprise one or more of the same components. For example, an amplification module may also comprise an input module, an output module, or both. In other examples, a system comprising a processor may be included in both the input module and the output module. A user may use the system to request that a target nucleic acid be amplified and may also be used to transmit information regarding amplified product to a recipient. In some cases, a system comprising a processor is included in all three modules, such that the system comprising a processor may also be used to control, provide instructions to, and receive information back from components included in the amplification module or any other module.

FIG. 1A and 1B provide an example of a system of the present disclosure. The system 100 may comprise a base 107 in the bottom. Within the base 107, there may be a source of excitation energy (e.g., a light source) 106 to direct energy to a sample comprised in a reaction vessel 104, thereby generating a signal from any amplification product present in the reaction vessel 104. The base 107 may also comprise a power supply (e.g., a battery) and/or a detector. The system may also comprise a heating member (e.g., a heating block) 105 positioned on the base 107 and in thermal communication with the reaction vessel 104. Power may be supplied to the heating member 105 by the power supply comprised in the base 107. The system may also comprise a housing 102 removably mountable to the base 107. The housing 102 may encapsulate the reaction vessel 104 when mounted to the base 107. The housing 102 may include at least one outlet 103 that permits flow of a convective fluid from a source of the convective fluid (e.g., generated by a fluid flow member 101, such as a fan) across the reaction vessel 104 to the at least one outlet 103. The housing 102 may also comprise a touch spot 108 that may function as a switch. For example, upon mounting of the housing 102 to the base 107, the touch spot 108 may be in contact with the base 107, thereby switching on the power supply. The power supply will then activate the heating member 105 (e.g., through a controller) to increase the temperature of reaction mixtures contained in the reaction vessel 104, which is in thermal communication with the heating member 105. When the temperature in the reaction vessel 104 reaches a first temperature level, heating may be stopped (e.g., by deactivating the heating member 105) , and the fluid flow member 101 may be activated to generate a flow of a convective fluid, so that it flows from a source thereof across the reaction vessel 104 to the at least one outlet 103, thereby lowering the temperature in the reaction vessel 104. When the temperature in the reaction vessel 104 reaches a second temperature level, the fluid flow member 101 may be deactivated, and one heating-cooling cycle may be completed. The cycles may be repeated for as many times as necessary.

FIG. 2 provides an example of a system of the present disclosure. Exterior appearance of a system 200 is shown. The system 200 may comprise a base 202, and a housing 205 mounted to the base 202. The housing 205 may comprise at least one out let 203, a switch button 204 and a display screen 201. When desired, the switch button 204 and/or the display screen 201 may be comprised by the base 202.

FIG. 3 shows internal structure of a system of the present disclosure 300. The system 300 may comprise a base 305. A fluid flow member 301 may be mounted on the base 305, and a heating member 303 may be mounted on the base 305 in parallel with the fluid flow member 301. A sample holder 302 may be held by a supporting member 307 mounted on the base 305 in the proximity of the heating member 303, so that the sample holder 302 and any reaction vessel secured by the sample holder will be in thermal communication with the heating member 303. The system may further comprise a controller (e.g., a Printed Circuit Board) 304 operatively connected to the heating member 303 and/or the fluid flow member 301. The controller 304 may be activated and/or deactivated by a switch 306 comprised in the system and operatively connected to the controller 304.

FIG. 4 provides a bottom view of an internal structure of a system of the present disclosure 400. The system may comprise a front cover 401, which may contain arrays of openings 402 to permit fluid flow (e.g., air flow) into and out of the system. The system may further comprise a power supply 403 (e.g., a battery) to supply power to the heating member 404 and/or the fluid flow member 405.

FIG. 5A demonstrates a display 501 that may be comprised in a system of the present disclosure. FIG. 5B to 5D provide examples of interfaces that may be shown on the display 501. FIG. 5B shows examples of the manner in which a temperature (e.g., a real-time temperature of a reaction mixture comprised in the reaction vessel) may be displayed. FIG. 5C and FIG. 5D shows examples of the manner in which a negative (FIG. 5C) or positive (FIG. 5D) amplification result may be displayed.

FIG. 6 provides an example of a reaction vessel 600 of the present disclosure. The reaction vessel 600 may comprise a cap 605 and a body 606. The body 606 may comprise a reaction chamber 604 and a sampling chamber 603 in fluid communication with the reaction chamber 604. The sampling chamber 603 may comprise an opening 602, and a collection member 601 comprised by the cap 605 may fit into the opening 602 when the cap is closed.

FIG. 7 shows a cross-sectional view of a reaction vessel 700 of the present disclosure. The reaction vessel 700 may comprise a reaction chamber 705 and a sampling chamber 703, there may be a seal 704 that partially or completely separates the reaction vessel 705 from the sampling vessel 703. There may be a sealing member 702 that seals an opening of the sampling chamber and prevents the contents therein from being exposed to the external environment. The reaction vessel 700 may also comprise a cap with a collection member 701, which may fit into the opening of the sampling chamber and penetrate the sealing member 702.

FIG. 8 shows a bottom view of a reaction vessel of the present disclosure. The reaction vessel may comprise a body and a cap 801, and the body may comprise a reaction chamber 803 and a sampling chamber 802. The bottom side 804 of the reaction vessel may be a layer with a thickness no more than about 0.3 mm.

Computer control systems

The present disclosure provides computer control systems that are programmed to implement methods of the disclosure. FIG. 9 shows a computer system 901 that is programmed or otherwise configured for sample processing and analysis, such as droplet generation and nucleic acid amplification and detection. The computer system 901 can regulate various aspects of methods and systems of the present disclosure.

The computer system 901 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 905, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 901 also includes memory or memory location 910 (e.g., random-access memory, read-only memory, flash memory) , electronic storage unit 915 (e.g., hard disk) , communication interface 920 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 925, such as cache, other memory, data storage and/or electronic display adapters. The memory 910, storage unit 915, interface 920 and peripheral devices 925 are in communication with the CPU 905 through a communication bus (solid lines) , such as a motherboard. The storage unit 915 can be a data storage unit (or data repository) for storing data. The computer system 901 can be operatively coupled to a computer network ( “network” ) 930 with the aid of the communication interface 920. The network 930 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 930 in some cases is a telecommunication and/or data network. The network 930 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 930, in some cases with the aid of the computer system 901, can implement a peer-to-peer network, which may enable devices coupled to the computer system 901 to behave as a client or a server.

The CPU 905 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 910. The instructions can be directed to the CPU 905, which can subsequently program or otherwise configure the CPU 905 to implement methods of the present disclosure. Examples of operations performed by the CPU 905 can include fetch, decode, execute, and writeback.

The CPU 905 can be part of a circuit, such as an integrated circuit. One or more other components of the system 901 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC) .

The storage unit 915 can store files, such as drivers, libraries and saved programs. The storage unit 915 can store user data, e.g., user preferences and user programs. The computer system 901 in some cases can include one or more additional data storage units that are external to the computer system 901, such as located on a remote server that is in communication with the computer system 901 through an intranet or the Internet.

The computer system 901 can communicate with one or more remote computer systems through the network 930. For instance, the computer system 901 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC) , slate or tablet PC’s (e.g.,

iPad,

Galaxy Tab) , telephones, Smart phones (e.g.,

iPhone, Android-enabled device,

) , or personal digital assistants. The user can access the computer system 901 via the network 930.

Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 901, such as, for example, on the memory 910 or electronic storage unit 915. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 905. In some cases, the code can be retrieved from the storage unit 915 and stored on the memory 910 for ready access by the processor 905. In some situations, the electronic storage unit 915 can be precluded, and machine-executable instructions are stored on memory 910.

The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

In another aspect, the present disclosure provides a non-transitory computer-readable medium comprising machine executable code that, upon execution by one or more computer processors, implements a method for conducting a chemical or biological reaction on a nucleic acid sample. The method may comprise activating a system comprising (i) a base comprising a heating member, wherein the base has a footprint that is less than or equal to about 5000 mm², (ii) a reaction vessel comprising a reaction chamber, wherein the reaction chamber is adjacent to and in thermal communication with the heating member. The reaction chamber may comprise a reaction mixture comprising a nucleic acid sample and reagents necessary for nucleic acid amplification. The system may further comprise (iii) a housing removably mounted to the base. The housing may encapsulate the reaction vessel, and the housing may include at least one outlet that permits flow of a convective fluid from a source of the convective fluid across the reaction chamber to the at least one outlet. The system may comprise (iv) a fluid flow member that subjects the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet. The method may further comprise subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture, by (i) directing the heating member to provide thermal energy to the reaction chamber, and (ii) directing the fluid flow member to subject the convective fluid to flow from the source of the convective fluid across the reaction chamber to the at least one outlet.

In another aspect, the present disclosure provides a non-transitory computer-readable medium comprising machine executable code that, upon execution by one or more computer processors, implements a method for nucleic acid amplification. The method may comprise activating a system comprising (i) a base comprising a heating member, the base may have a footprint that is less than or equal to about 5000 mm², (ii) a reaction vessel comprising a reaction chamber, the reaction chamber may be adjacent to and in thermal communication with the heating member, and the reaction chamber may comprise a reaction mixture comprising a nucleic acid sample and reagents necessary for nucleic acid amplification, and (iii) a housing removably mounted to the base, the housing may encapsulate the reaction vessel, and the housing may include a cooling member that is in thermal communication with the reaction vessel when the housing is mounted to the base. The method may further comprise subjecting the reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on the reaction mixture, this may be achieved by regulating (i) a rate of heating of the reaction vessel using the heating member and (ii) a rate of cooling of the reaction vessel using the cooling member.

Aspects of the systems and methods provided herein, such as the computer system 901, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer (s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables； copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The computer system 901 can include or be in communication with an electronic display 935 that comprises a user interface (UI) 940 for providing, for example, nucleic acid sequence information. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 905. The algorithm can, for example, regulate systems or implement methods provided herein.

Devices, systems and methods of the present disclosure may be combined with other devices, systems or methods, such as those described in PCT/CN14/094914 and PCT/CN14/078022, each of which is entirely incorporated herein by reference.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A system for nucleic acid amplification, comprising:

a base comprising a heating member, wherein said base has a footprint that is less than or equal to about 5000 mm²；

a reaction vessel comprising a reaction chamber, wherein said reaction chamber is adjacent to and in thermal communication with said heating member, wherein during use, said heating member provides thermal energy to a reaction mixture in said reaction chamber, which reaction mixture comprises a nucleic acid sample and reagents necessary for nucleic acid amplification；

a housing removably mountable to said base, wherein said housing encapsulates said reaction vessel when mounted to said base, and wherein said housing includes at least one outlet that permits flow of a convective fluid from a source of said convective fluid across said reaction chamber to said at least one outlet；

a fluid flow member that subjects said convective fluid to flow from said source of said convective fluid across said reaction chamber to said at least one outlet； and

a controller operatively coupled to said heating member and said fluid flow member, wherein said controller is programmed to subject said reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on said reaction mixture, by (i) directing said heating member to provide thermal energy to said reaction chamber, and (ii) directing said fluid flow member to subject said convective fluid to flow from said source of said convective fluid across said reaction chamber to said at least one outlet.
The system of claim 1, wherein said reaction chamber has a footprint that is less than or equal to about 1000 mm².
The system of claim 2, wherein said footprint is less than or equal to about 500 mm².
The system of claim 3, wherein said footprint is less than or equal to about 300 mm².
The system of claim 1, wherein said base has a footprint that is less than or equal to about 2000 mm².
The system of claim 1, wherein said housing mounted to said base has a footprint that is less than or equal to about 5000 mm².
The system of claim 1, wherein a cross-section of said reaction chamber is less than a cross-section of said heating member.
The system of claim 1, wherein said reaction chamber has a surface area to volume ratio of at least 100 mm^-1.
The system of claim 1, further comprising a power supply that supplies power to said heating member.
The system of claim 9, wherein said power supply supplies power to said fluid flow member.
The system of claim 9, further comprising a switch in said base or said housing operatively connected to said power supply, wherein said switch regulates supply of power from said power supply to said heating member.
The system of claim 11, wherein said switch directs supply of power from said power supply to said heating member when said housing is mounted to said base.
The system of claim 1, wherein said housing comprises a chamber that encapsulates said reaction vessel when mounted to said base.
The system of claim 1, further comprising a sample holder mounted to said heating member, which sample holder receives and secures said reaction vessel.
The system of claim 14, wherein said reaction chamber is adjacent to and in thermal communication with said heating member when said reaction vessel is secured to said sample holder.
The system of claim 1, wherein said base further comprises an excitation energy source operatively coupled to said reaction vessel, wherein said excitation energy source provides excitation energy to said reaction vessel to induce a signal (s) that is indicative of a presence or absence of an amplification product of said nucleic acid amplification reaction.
The system of claim 16, further comprising a first sensor in sensing communication with said reaction vessel, wherein said first sensor detects said signal (s) that is indicative of said presence or absence of said amplification product.
The system of claim 17, wherein said first sensor is an optical sensor, and wherein said signal (s) is an optical signal.
The system of claim 1 or 17, further comprising a second sensor in sensing communication with said reaction vessel, wherein said second sensor detects a temperature at one or more location (s) within said reaction chamber of said reaction vessel.
The system of claim 17, further comprising a display in said base or said housing operatively coupled to said first sensor, wherein said display is configured to display said signal (s) indicative of said presence or absence of said amplification product.
The system of claim 19, further comprising a display in said base or said housing operatively coupled to said first sensor and/or said second sensor, wherein said display is configured to:

i) display said signal (s) indicative of said presence or absence of said amplification product, and/or

2) display said temperature at said one or more location (s) within said reaction chamber of said reaction vessel.
The system of claim 16, wherein said excitation energy source is a light source.
The system of claim 1, wherein said convective fluid is a convective gas.
The system of claim 23, wherein said convective gas is air.
The system of claim 1, wherein said fluid flow member is a fan.
The system of claim 1, wherein said fluid flow member is mounted to said housing or to said base.
The system of claim 1, wherein said source of said convective fluid is a refrigeration unit.
The system of claim 1, wherein said heating member includes an infrared heating unit.
The system of claim 1, wherein said heating member includes a Peltier heating unit.
The system of claim 1, wherein said heating member includes an aluminum-containing heating unit.
The system of claim 1, wherein said heating member includes an electrically resistive heating unit.
The system of claim 1, wherein said controller is programmed to direct said heating member to provide thermal energy to said reaction chamber until said reaction mixture reaches a first temperature, and direct said fluid flow member to subject said convective fluid to flow across said reaction chamber until said reaction mixture reaches a second temperature that is less than said first temperature.
The system of claim 32, wherein said controller is programmed to direct said fluid flow member to reduce or terminate flow of said convective fluid when said reaction mixture reaches said second temperature.
The system of claim 1, wherein said controller is included in said base.
The system of claim 1, wherein said reaction mixture includes one or more primers and polymerizing enzymes.
The system of claim 35, wherein said reaction mixture includes a buffer.
The system of claim 35, wherein said reaction mixture includes cations that regulate an activity of said polymerizing enzymes.
The system of claim 37, wherein said cations include Mg²⁺ or Mn²⁺.
The system of claim 35, wherein said one or more primers have nucleic acid sequences that are selected for HBV, HCV, FluA, FluB, CA16, EV71, enterovirus, EBOV, EBV, measles virus, salmonella, HPV and/or HIV.
The system of claim 1, wherein said nucleic acid amplification reaction is polymerase chain reaction (PCR) .
The system of claim 1, wherein said convective fluid is at an average temperature of less than about 15℃.
The system of claim 41, wherein said convective fluid is at an average temperature of less than about 10℃.
The system of claim 1, wherein said controller provides heating and/or cooling to said reaction mixture by controlling a heating rate of said reaction chamber using said thermal energy provided by said heating member and a cooling rate of said reaction chamber using said flow of said convective fluid across said reaction chamber.
The system of claim 43, wherein said reaction mixture is subjected to heating when said heating rate is greater than said cooling rate.
The system of claim 43, wherein said reaction mixture is subjected to cooling when said heating rate is less than said cooling rate.
The system of claim 43, wherein said heating rate is at least 5℃/s.
The system of claim 43, wherein said cooling rate is at least 5℃/s.
The system of claim 1, wherein said controller does not subject said reaction mixture to said one or more cycles of heating and cooling in the absence of said switch being turned on.
The system of claim 1, wherein said controller subjects said reaction mixture to said one or more cycles of heating and cooling after a time delay upon said switch being turned on.
The system of claim 1, wherein said reaction vessel further comprises a sampling unit, and wherein said sampling unit comprises a sampling chamber in fluid communication with said reaction chamber.
The system of claim 50, wherein said sampling unit further comprises a collection member that collects a nucleic acid sample.
The system of claim 51, wherein said sampling unit further comprises a sealing member that seals an opening of said sampling chamber.
The system of claim 52, wherein, during use, said collection member pierces said sealing member to release said nucleic acid sample into said sampling chamber.
The system of claim 1, wherein a side of said reaction chamber adjacent to said heating member has a thickness of less than about 1 mm.
A method for nucleic acid amplification, comprising:

(a) activating a system comprising (i) a base comprising a heating member, wherein said base has a footprint that is less than or equal to about 5000 mm², (ii) a reaction vessel comprising a reaction chamber, wherein said reaction chamber is adjacent to and in thermal communication with said heating member, wherein said reaction chamber comprises a reaction mixture comprising a nucleic acid sample and reagents necessary for nucleic acid amplification, (iii) a housing removably mounted to said base, wherein said housing encapsulates said reaction vessel, wherein said housing includes at least one outlet that permits flow of a convective fluid from a source of said convective fluid across said reaction chamber to said at least one outlet, and (iv) a fluid flow member that subjects said convective fluid to flow from said source of said convective fluid across said reaction chamber to said at least one outlet； and

(b) subjecting said reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on said reaction mixture, by (i) directing said heating member to provide thermal energy to said reaction chamber, and (ii) directing said fluid flow member to subject said convective fluid to flow from said source of said convective fluid across said reaction chamber to said at least one outlet.
The method of claim 55, wherein said reaction chamber has a footprint that is less than or equal to about 1000 mm².
The method of claim 55, wherein said base has a footprint that is less than or equal to about 2000 mm².
The method of claim 55, wherein said housing mounted to said base has a footprint that is less than or equal to about 5000 mm².
The method of claim 55, wherein a cross-section of said reaction chamber is less than a cross-section of said heating member.
The method of claim 55, wherein said reaction chamber has a surface area to volume ratio of at least 100 mm^-1.
The method of claim 55, wherein said activating in (a) is by supplying power to said heating member with a power supply.
The method of claim 61, wherein said power supply supplies power to said fluid flow member.
The method of claim 61, wherein said power is supplied by turning on a switch operatively connected to said power supply in said base or said housing.
The method of claim 63, wherein said switch is turned on by mounting said housing to said base.
The method of claim 55, wherein said housing comprises a chamber that encapsulates said reaction vessel when mounted to said base.
The method of claim 55, further comprising, prior to (a) , securing said reaction vessel to a sample holder mounted to said heating member.
The method of claim 66, wherein said reaction chamber is adjacent to and in thermal communication with said heating member when said reaction vessel is secured to said sample holder.
The method of claim 55, further comprising providing excitation energy to said reaction vessel to induce a signal (s) that is indicative of a presence or absence of an amplification product of said nucleic acid amplification reaction.
The method of claim 68, further comprising providing a first sensor in sensing communication with said reaction vessel to detect said signal (s) that is indicative of said presence or absence of said amplification product.
The method of claim 69, wherein said first sensor is an optical sensor, and wherein said signal (s) is an optical signal.
The method of claim 69, further comprising providing a second sensor in sensing communication with said reaction vessel to detect a temperature at one or more location (s) within said reaction chamber of said reaction vessel.
The method of claim 71, further comprising providing a display operatively coupled to said first sensor and/or said second sensor in said base or said housing, wherein said display is configured to:(1) display said signal (s) indicative of said presence or absence of said amplification product, and/or (2) display said temperature at said one or more location (s) within said reaction chamber of said reaction vessel.
The method of claim 69, further comprising providing a display operatively coupled to said first sensor in said base or said housing, wherein said display is configured to display said signal (s) indicative of said presence or absence of said amplification product.
The method of claim 68, wherein said excitation energy source is a light source.
The method of claim 55, wherein said convective fluid is a convective gas.
The method of claim 75, wherein said convective gas is air.
The method of claim 55, wherein said fluid flow member is a fan.
The method of claim 55, wherein said fluid flow member is mounted to said housing or to said base.
The method of claim 55, wherein said source of said convective fluid is a refrigeration unit.
The method of claim 55, further comprising, prior to (a) , depositing said base having said housing mounted thereto in a refrigeration unit.
The method of claim 55, wherein said heating member includes an infrared heating unit.
The method of claim 55, wherein said heating member includes a Peltier heating unit.
The method of claim 55, wherein said heating member includes an aluminum-containing heating unit.
The method of claim 55, wherein said heating member includes an electrically resistive heating unit.
The method of claim 55, wherein thermal energy is provided to said reaction chamber until said reaction mixture reaches a first temperature, and wherein said convective fluid is subjected to flow across said reaction chamber until said reaction mixture reaches a second temperature that is less than said first temperature.
The method of claim 85, further comprising reducing or terminating flow of said convective fluid when said reaction mixture reaches said second temperature.
The method of claim 55, wherein said reaction mixture includes one or more primers and polymerizing enzymes.
The method of claim 87, wherein said reaction mixture includes a buffer.
The method of claim 87, wherein said reaction mixture includes cations that regulate an activity of said polymerizing enzymes.
The method of claim 89, wherein said cations include Mg²⁺ or Mn²⁺.
The method of claim 87, wherein said one or more primers have nucleic acid sequences that are selected for HBV, HCV, FluA, FluB, CA16, EV71, enterovirus, EBOV, EBV, measles virus, salmonella, HPV and/or HIV.
The method of claim 55, wherein said nucleic acid amplification reaction is polymerase chain reaction (PCR) .
The method of claim 55, further comprising controlling a heating rate of said reaction chamber using said thermal energy provided by said heating member and a cooling rate of said reaction chamber using said flow of said convective fluid across said reaction chamber.
The method of claim 93, wherein said heating rate is greater than said cooling rate, thereby subjecting said reaction mixture to heating.
The method of claim 93, wherein said heating rate is less than said cooling rate, thereby subjecting said reaction mixture to cooling.
The method of claim 93, wherein said heating rate is at least 5℃/s.
The method of claim 93, wherein said cooling rate is at least 5℃/s.
The method of claim 55, wherein in (b) , said reaction mixture is not subjected to said one or more cycles of heating and cooling in the absence of said switch being turned on.
The method of claim 55, wherein in (b) , said reaction mixture is subjected to said one or more cycles of heating and cooling after a time delay upon said switch being turned on.
The method of claim 55, further comprising, subsequent to (b) , deactivating said system.
The method of claim 55, further comprising, prior to (a) , depositing said nucleic acid sample in said reaction vessel.
The method of claim 101, wherein said reaction vessel further comprises a sampling unit, and wherein said sampling unit comprises a collection member and a sampling chamber in fluid communication with said reaction chamber, wherein said nucleic acid sample is deposited in said reaction vessel by piercing a sealing member sealing an opening of said sampling chamber with said collection member having collected said nucleic acid sample thereon or therein.
A system for nucleic acid amplification, comprising:

a base comprising a heating member, wherein said base has a footprint that is less than or equal to about 5000 mm²；

a reaction vessel comprising a reaction chamber, wherein said reaction chamber is adjacent to and in thermal communication with said heating member, wherein during use, said heating member provides thermal energy to a reaction mixture in said reaction chamber, which reaction mixture comprises a nucleic acid sample and reagents necessary for nucleic acid amplification；

a housing removably mountable to said base, wherein said housing encapsulates said reaction vessel when mounted to said base, and wherein said housing includes a cooling member that is in thermal communication with said reaction vessel when said housing is mounted to said base；

a controller operatively coupled to said heating member, wherein said controller is programmed to subject said reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on said reaction mixture, by regulating (i) a rate of heating of said reaction vessel using said heating member and (ii) a rate of cooling of said reaction vessel using said cooling member.
The system of claim 103, wherein said cooling member is formed of a material with a heat capacity of at least about 0.2 J/g*K.
The system of claim 104, wherein said cooling member is formed of a material with a heat capacity of at least about 0.3 J/g*K.
The system of claim 105, wherein said cooling member is formed of a material with a heat capacity of at least about 0.4 J/g*K.
The system of claim 106, wherein said cooling member is formed of a material with a heat capacity of at least about 0.5 J/g*K.
The system of claim 107, wherein said cooling member is formed of a material with a heat capacity of at least about 1.0 J/g*K.
The system of claim 103, wherein said cooling member is a solid comprising copper.
A method for nucleic acid amplification, comprising:

(a) activating a system comprising (i) a base comprising a heating member, wherein said base has a footprint that is less than or equal to about 5000 mm², (ii) a reaction vessel comprising a reaction chamber, wherein said reaction chamber is adjacent to and in thermal communication with said heating member, wherein said reaction chamber comprises a reaction mixture comprising a nucleic acid sample and reagents necessary for nucleic acid amplification, and (iii) a housing removably mounted to said base, wherein said housing encapsulates said reaction vessel, wherein said housing includes a cooling member that is in thermal communication with said reaction vessel when said housing is mounted to said base； and

(b) subjecting said reaction mixture to one or more cycles of heating and cooling to facilitate a nucleic acid amplification reaction on said reaction mixture, by regulating (i) a rate of heating of said reaction vessel using said heating member and (ii) a rate of cooling of said reaction vessel using said cooling member.
The method of claim 110, wherein said reaction chamber has a footprint that is less than or equal to about 1000 mm².
The method of claim 110, wherein said base has a footprint that is less than or equal to about 2000 mm².
The method of claim 110, wherein said housing mounted to said base has a footprint that is less than or equal to about 5000 mm².
The method of claim 110, further comprising providing excitation energy to said reaction vessel to induce a signal (s) that is indicative of a presence or absence of an amplification product of said nucleic acid amplification reaction.
The method of claim 114, further comprising providing a first sensor in sensing communication with said reaction vessel to detect said signal (s) that is indicative of said presence or absence of said amplification product.
The method of claim 115, wherein said first sensor is an optical sensor, and wherein said signal (s) is an optical signal.
The method of claim 115, further comprising providing a second sensor in sensing communication with said reaction vessel to detect a temperature at one or more location (s) within said reaction chamber of said reaction vessel.
The method of claim 117, further comprising providing a display operatively coupled to said first sensor and/or said second sensor in said base or said housing, wherein said display is configured to:(1) display said signal (s) indicative of said presence or absence of said amplification product, and/or (2) display said temperature at said one or more location (s) within said reaction chamber of said reaction vessel.
The method of claim 115, further comprising providing a display operatively coupled to said first sensor in said base or said housing, wherein said display is configured to display said signal (s) indicative of said presence or absence of said amplification product.