CN101812543A - Kit for detecting human immunodeficiency virus - Google Patents
Kit for detecting human immunodeficiency virus Download PDFInfo
- Publication number
- CN101812543A CN101812543A CN201010164037A CN201010164037A CN101812543A CN 101812543 A CN101812543 A CN 101812543A CN 201010164037 A CN201010164037 A CN 201010164037A CN 201010164037 A CN201010164037 A CN 201010164037A CN 101812543 A CN101812543 A CN 101812543A
- Authority
- CN
- China
- Prior art keywords
- mediated isothermal
- gold particles
- loop
- dna
- hiv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000725303 Human immunodeficiency virus Species 0.000 title claims abstract description 49
- 239000010931 gold Substances 0.000 claims abstract description 29
- 229910052737 gold Inorganic materials 0.000 claims abstract description 29
- 239000002245 particle Substances 0.000 claims abstract description 28
- 230000003321 amplification Effects 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 18
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 18
- 230000001404 mediated effect Effects 0.000 claims abstract description 12
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 12
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 12
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 230000008569 process Effects 0.000 claims abstract description 7
- 238000010839 reverse transcription Methods 0.000 claims abstract description 6
- 108020004414 DNA Proteins 0.000 claims description 27
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 27
- 239000000523 sample Substances 0.000 claims description 25
- 238000007397 LAMP assay Methods 0.000 claims description 20
- 238000012360 testing method Methods 0.000 claims description 11
- 108020005202 Viral DNA Proteins 0.000 claims description 10
- 241000700605 Viruses Species 0.000 claims description 10
- 239000003068 molecular probe Substances 0.000 claims description 9
- 108020000999 Viral RNA Proteins 0.000 claims description 8
- 238000009396 hybridization Methods 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 3
- 238000011901 isothermal amplification Methods 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims 2
- 101001007348 Arachis hypogaea Galactose-binding lectin Proteins 0.000 claims 1
- 239000000470 constituent Substances 0.000 claims 1
- 230000007812 deficiency Effects 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 230000035484 reaction time Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000007689 inspection Methods 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 238000003759 clinical diagnosis Methods 0.000 abstract description 2
- 208000035473 Communicable disease Diseases 0.000 abstract 1
- 241000588724 Escherichia coli Species 0.000 abstract 1
- 238000003745 diagnosis Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 8
- 206010061598 Immunodeficiency Diseases 0.000 description 7
- 208000029462 Immunodeficiency disease Diseases 0.000 description 7
- 230000007813 immunodeficiency Effects 0.000 description 7
- 208000030507 AIDS Diseases 0.000 description 5
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001104043 Syringa Species 0.000 description 1
- 235000004338 Syringa vulgaris Nutrition 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kit for detecting product DNA by utilizing nano-gold particles after human immunodeficiency virus is amplified through a loop-mediated isothermal nucleic acid amplification technique, which belongs to the technical field of biological detection. The kit combines the loop-mediated isothermal nucleic acid amplification technique with a biological nanotechnology to improve the detection sensitivity of biological molecules, is a method with simple operation, rapidness, accuracy and no need for professional instruments and equipment, and can be widely applied to high-sensitivity HIV detection in the fields of household diagnosis, clinical diagnosis, infectious diseases control, environment monitoring, inspection and quarantine, biotechnology and the like. The kit comprises two parts, namely (1) the nano-gold particles marked with molecular robes capable of identifying specific sequences of HIV and (2) a loop-mediated isothermal nucleic acid amplification system capable of amplifying the escherichia coli DNA in to-be-detected samples or performing a reverse transcription process and then amplifying the RNA of the HIV in the to-be-detected samples.
Description
One, technical field
The present invention relates to a kind ofly detect the test kit of human immunodeficiency virus (HIV), belong to technical field of biological through loop-mediated isothermal amplification amplification after product DNA with nm gold particles.
Two, background technology
To the high sensitivity of gene (DNA) and transcription product (RNA) thereof, high specific, simply, detect fast, for the early diagnosis and therapy of disease and all have crucial meaning in fields such as health and epidemic prevention, environmental monitoring, inspection and quarantine.Comparatively accurate in this respect in the past sensitive detection method has methods such as fluorescent marker method, radio-labeling, and these methods all need complicated operations and special equipment, and range of application is very narrow.The Dr.Kary B.Mullis of PE company in 1993 has invented polymerase chain reaction (Polymerase Chain Reaction), is called for short PCR.Round pcr is applied in biological scientific research and the clinical treatment on a large scale subsequently, and the detection of DNA has been entered the new epoch, and Dr.Mullis has also obtained Nobel chemistry Prize in 1993 for this reason.Though advantages such as PCR method has fast, sensitivity, accuracy is high, operation is simpler are because its reaction is carried out needing special instrument, the use of round pcr to be limited in the laboratory and hospital of specialty with the detection of product dna always.
Calendar year 2001, Japan Eiken Chemical developed a kind of Novel DNA amplification mode, loop-mediated isothermal amplification (LAMP) can be realized the efficient rapid amplifying of DNA under constant temperature, this method reaction conditions is simple, therefore DNA cloning efficient height has clear superiority aspect detection of nucleic acids.But the DNA product to LAMP amplification preparation uses conventional detection method, and as electrophoretic method, fluorescence dye method etc. all can't be got rid of the false positive problem that the LAMP amplification is brought, and need special instrument, have therefore limited the practical application of LAMP technology.
(Human Immunodeficiency Virus HIV), can cause the defective of human immunity system to human immunodeficiency virus.1981, human immunodeficiency virus was found first in the U.S..It is a kind of slow virus of infected person para-immunity system cells, belongs to a kind of of retrovirus.So far the mortality transmissible disease of not having effective therapy.The immunological competence of this virus damage human body causes the immune resistibility that loses, and causes various diseases and cancer to be able to survive in human body, develops at last, causes acquired immune deficiency syndrome (AIDS) (AIDS: acquired immune deficiency syndrome (AIDS)).Lack detection method simple, quick, easy to use and with low cost at HIV virus at present.
Three, summary of the invention
The objective of the invention is to propose a kind of with the test kit of nm gold particles detection HIV virus through loop-mediated isothermal amplification amplification after product DNA, coupling collar mediated isothermal amplification and biological nano technology, improve the sensitivity of biomolecule detection, it is a kind of simple to operate, quick, accurate, with low cost, method of not needing professional plant and instrument, can be widely used in home diagnostic, clinical diagnosis, transmissible disease control, environmental monitoring, inspection and quarantine, the highly sensitive HIV virus of biotechnology field detects.
Test kit of the present invention comprises two portions:
1. be marked with the nm gold particles of the molecular probe that can discern HIV virus particular sequence;
2. can increase in the detected sample the HIV viral DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the HIV viral RNA in the detected sample that increases again after the reverse transcription process.
The principle of work of this test kit is:
1. nm gold particles is marked with the molecular probe that can discern the HIV particular sequence, and molecular probe is fixed on the surface of nm gold particles.
2. HIV viral DNA molecule in the detected sample or HIV viral RNA are through loop-mediated isothermal amplification massive duplication or first massive duplication after the reverse transcription process, and the target fragment of HIV virus is exaggerated 10
9Doubly, the amplified production DNA of generation has multiple tumor-necrosis factor glycoproteins.
3. mix nm gold particles and ring mediated isothermal nucleic acid amplification system, HIV virus can be discerned the molecular probe hybridization of HIV virus particular sequence on product D NA after the loop-mediated isothermal amplification and nm gold particles.
4. nm gold particles is along product of loop-mediated isothermal amplification dna molecular chain formation aggregate (as shown in drawings), and then cause the colour-change of nm gold particles habitat (for example in the solution, solid surface etc.), can judge according to colour-change whether testing sample has HIV viral DNA or RNA.
Four, description of drawings
Be marked with the nm gold particles and the product D NA hybridization synoptic diagram of HIV virus after loop-mediated isothermal amplification of molecular probe, nm gold particles is along product of loop-mediated isothermal amplification dna molecular chain formation aggregate.
Five, embodiment
Test kit of the present invention comprises 2 pipe solution, be the nm gold particles solution 50 μ l that are marked with the molecular probe that can discern HIV virus particular sequence in one pipe, in the pipe be 250 μ l can increase in the detected sample the HIV viral DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the HIV viral RNA in the detected sample that increases again after the reverse transcription process.
Contain the nm gold particles that diameter is 20nm in the nm gold particles solution, concentration is 1.2nM, and the nm gold particles surface markers has the dna probe of sulfydryl modification.
Ring mediated isothermal nucleic acid amplification system composition is as follows: 0.2 μ M F3 and B3 primer, 1.6 μ M FIP and BIP primer, 0.8 μ M LoopF and LoopB primer, 0.4M betaine (Sigma-Aldrich, St.Louis, MO), 10mM MgSO4,1.4mM dNTPs, 1 * ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 80U Bst DNApolymerase (New England Biolabs), 6.25U AMV reversed transcriptive enzyme (Invitrogen, Carlsbad, CA).
The dna probe sequence of sulfydryl modification
5′-AGTGGCACCAGGCCAGAAAAAAAA-SH-3′
Primer sequence
The FIP primer of HIV
5′-TTTAACATTTGCATGGCTGCT-TACCCATGTTTACAGCATTATC-3′
The BIP primer of HIV
5′-ATGAGAGAACCAAGGGGAAGTGTCATGGATCCTATTTGCTCC-3′
The F3 primer of HIV
5′-AAGGCTTTTAGCCCAGAAG-3′
The B3 primer of HIV
5′-TCTCCTACTGGGATAGGTGG-3′
The LoopF primer of HIV
5′-TAAATCTTGTGGGGTGGCTC-3′
The LoopB primer of HIV
5′-CATAGCAGGAACTACTAGTACTCTT-3′
Operating process is as follows
1. in the pipe that ring mediated isothermal nucleic acid amplification system is housed, add nucleic acid or the blood sample that 10 μ l extract.
2. the pipe that mixture will be housed is put into 60 ℃ water-bath 30 minutes.
3. add nm gold particles solution in the pipe in step 2 again, this moment, mixed solution was red.
4.60 ℃ water-bath 15 minutes.
5. the color of solution in the pipe behind the observing response, solution keeps red constant specific RNA of HIV virus infection or the HIV viral DNA of then not containing; If solution becomes lilac, judge that then containing HIV virus in the sample dyes specific RNA or HIV viral DNA.In such cases, if sample is a blood sample, provide the people of blood sample to be infected so by HIV virus.
Sequence table
＜110〉Truehigh Tech Co., Ltd.
＜120〉a kind of test kit that detects human immunodeficiency virus
<130>Demo
<160>7
<170>PatentIn?version?3.5
<210>1
<211>24
<212>DNA
<213>Human?immunodeficiency?virus
<400>1
agtggcacca?ggccagaaaa?aaaa 24
<210>2
<211>43
<212>DNA
<213>Human?immunodeficiency?virus
<400>2
tttaacattt?gcatggctgc?ttacccatgt?ttacagcatt?atc?43
<210>3
<211>42
<212>DNA
<213>Human?immunodeficiency?virus
<400>3
atgagagaac?caaggggaag?tgtcatggat?cctatttgct?cc 42
<210>4
<211>19
<212>DNA
<213>Human?immunodeficiency?virus
<400>4
aaggctttta?gcccagaag 19
<210>5
<211>20
<212>DNA
<213>Human?immunodeficiency?virus
<400>5
tctcctactg?ggataggtgg 20
<210>6
<211>20
<212>DNA
<213>Human?immunodeficiency?virus
<400>6
taaatcttgt?ggggtggctc 20
<210>7
<211>25
<212>DNA
<213>Human?immunodeficiency?virus
<400>7
catagcagga?actactagta?ctctt 25
Claims (8)
1. one kind is detected human immune deficiency (HIV) the virus test kit through loop-mediated isothermal amplification amplification after product DNA with nm gold particles, and it is characterized in that this test kit comprises with the lower section: (1) is marked with the nm gold particles of the molecular probe that can discern HIV virus particular sequence; (2) can increase in the detected sample the HIV viral DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the HIV viral RNA in the detected sample that increases again after the reverse transcription process.
2. according to claim 1, the principle of work of this test kit be (1) with the HIV viral DNA in the loop-mediated isothermal amplification amplification detected sample or earlier through after the reverse transcription process with the HIV viral RNA in the loop-mediated isothermal amplification amplification detected sample; (2) nm gold particles probe and product of loop-mediated isothermal amplification DNA hybridization; (3) hybridization causes the nm gold particles gathering, causes nm gold particles habitat color change, judges with this whether detected sample contains HIV viral DNA or RNA
3. according to claim 1, the nm gold particles probe comprises two parts, the one, and the gold grain of nanoscale, another part are the molecular probes that is fixed on the nm gold particles surface, this probe can be discerned the particular sequence of HIV virus.
4. according to claim 2, the amplification object of loop-mediated isothermal amplification can be the mixture of HIV viral DNA, RNA or DNA and RNA in the detected sample.
5. according to claim 2, nm gold particles probe and product of loop-mediated isothermal amplification DNA hybridization can be carried out under same reaction conditions, in the same reaction container simultaneously with the ring mediated isothermal nucleic acid amplification reaction.
6. according to claim 2, nm gold particles probe and product of loop-mediated isothermal amplification DNA hybridization can occur in the solution or solid surface.
7. according to claim 2, nm gold particles probe and product of loop-mediated isothermal amplification DNA hybridization cause solution or solid surface color change, and this variation can directly be judged by naked eyes, also can utilize opticinstrument detections such as spectrophotometer.
9. according to claim 3, molecular probe be meant can identification ring mediated isothermal amplification product D NA functional molecular, these molecules comprise DNA, RNA, PNA, protein, chemical molecular.
9. according to claim 4, same reaction conditions includes but not limited to: solution composition, constituent concentration, pH value, temperature, reaction times etc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010164037A CN101812543A (en) | 2010-05-06 | 2010-05-06 | Kit for detecting human immunodeficiency virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010164037A CN101812543A (en) | 2010-05-06 | 2010-05-06 | Kit for detecting human immunodeficiency virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101812543A true CN101812543A (en) | 2010-08-25 |
Family
ID=42619911
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010164037A Pending CN101812543A (en) | 2010-05-06 | 2010-05-06 | Kit for detecting human immunodeficiency virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101812543A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567867A (en) * | 2014-10-11 | 2016-05-11 | 上海仁度生物科技有限公司 | Human immunodeficiency virus 1 real-time fluorescent nucleic acid isothermal amplification detection kit |
| CN115494135A (en) * | 2022-09-05 | 2022-12-20 | 青岛科技大学 | Based on COF film and AgInS 2 Photoelectrochemical biosensor of quantum dots and application of photoelectrochemical biosensor in detection of double targets |
-
2010
- 2010-05-06 CN CN201010164037A patent/CN101812543A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567867A (en) * | 2014-10-11 | 2016-05-11 | 上海仁度生物科技有限公司 | Human immunodeficiency virus 1 real-time fluorescent nucleic acid isothermal amplification detection kit |
| CN115494135A (en) * | 2022-09-05 | 2022-12-20 | 青岛科技大学 | Based on COF film and AgInS 2 Photoelectrochemical biosensor of quantum dots and application of photoelectrochemical biosensor in detection of double targets |
| CN115494135B (en) * | 2022-09-05 | 2024-10-01 | 青岛科技大学 | A photoelectrochemical biosensor based on COF film and AgInS2 quantum dots and its dual-target detection application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101659999B (en) | Method for detecting product of loop-mediated isothermal amplification | |
| Choi et al. | An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care | |
| CN104093856B (en) | Method for detecting nucleic acid synthesis and/or amplification | |
| EP3087202B1 (en) | Nucleic acid detection method and kit | |
| JPWO2017212904A1 (en) | Rapid detection method of African swine fever virus using LAMP method combining multiple primer sets | |
| CN105755134B (en) | Endonuclease-mediated real-time multiple cross-displacement nucleic acid amplification technology and application | |
| KR20210158368A (en) | A method of detecting rna | |
| CN115109859B (en) | A multimodal composite functional Helicobacter pylori nucleic acid detection test strip | |
| US20240141410A1 (en) | Detection of a target polynucleotide | |
| US12325882B2 (en) | Fast and portable microfluidic detection system as an alternative to Salmonella's classical culture method | |
| CN106893764A (en) | A kind of LAMP detection primer combination of Ralstonia solanacearum and its LAMP detection kit and LAMP method | |
| CN101812544A (en) | Influenza virus detection kit | |
| CN101812543A (en) | Kit for detecting human immunodeficiency virus | |
| CN102586439A (en) | Method for simultaneously and quickly detecting multiple nucleic acids | |
| KR101437385B1 (en) | Primer set for Brucella abortus strain-specific identification using loop-mediated isothermal amplification and method for detecting Brucella abortus using the same | |
| CN101824480A (en) | Kit for detecting Salmonella spp. | |
| CN101824492A (en) | Kit for detecting AH1N1 influenza virus | |
| CN101824491A (en) | Kit for detecting hepatitis C virus | |
| CN101812531A (en) | Escherichia coli detection kit | |
| CN118460747A (en) | Method and application of detecting Staphylococcus aureus based on Argonaute and centrifugal microfluidic chip | |
| CN101824493A (en) | Kit for detecting hepatitis B virus | |
| CN102876806A (en) | Kit for detecting H4N6 avian influenza virus | |
| Gooch et al. | Application of fluorescent substrates to the in situ detection of prostate specific antigen | |
| CN102230026A (en) | Human enterovirus 71 detection kit | |
| CN102226222A (en) | Kit for detecting coxsackie virus A16 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20100825 |