CN113930501A - Digital PCR detection method for human EGFR gene mutation and application - Google Patents

Abstract

The invention provides a digital PCR detection method and application of human EGFR gene mutation. Specifically, the preferred primer pairs, amplification methods, nucleic acid probes and detection systems for the sequences of EGFR S768, EGFR L861 and EGFR G719 are provided, and a kit for detecting EGFR gene mutation is further provided. The present invention can detect EGFR gene mutation with high sensitivity and strong specificity for different samples through optimized primer pairs, probes and corresponding reaction conditions.

Description

Digital PCR detection method and application of human EGFR gene mutation

technical field

The invention relates to the field of gene detection, in particular to a digital PCR detection method and application of human EGFR gene mutation.

Background technique

For patients with advanced non-small cell lung cancer with EGFR mutation, epidermal growth factor receptor-tyrosine inhibitors (EGFR-TKIs) targeted drugs are very effective, such as gefitinib, erlotinib (erlotinib), afatinib, dasatinib, and osimertinib.

EGFR mutations, especially exon 19 deletion, exon 21 (L858R, L861), exon 18 (G719X, G719) and exon 20 (S768I) mutations, are sensitive to EGFR-type tyrosine kinase inhibitors Sex is highly correlated. Therefore, detecting whether the EGFR gene is mutated becomes a prerequisite for the use of EGFR-targeted drugs in lung cancer patients.

Exon 19 deletion and L858R mutation are common mutations. For lung cancer patients with these two mutations, the first-generation EGFR inhibitors gefitinib, erlotinib and the second-generation EGFR inhibitor afatinib can be recommended for first-line use treat. The domestic drug icotinib is also recommended for first-line treatment in the CSCO guidelines.

Exon 19 deletion and L858R mutation are called rare mutations, such as L861Q, G719X, S768I, etc. These mutation sites are suitable for the use of the second-generation targeted drug afatinib.

Therefore, the detection of rare mutations of EGFR gene S768I, L861Q, G719X (including G719S, G719C, G719A) can provide reference for doctors to select targeted drug therapy in patients with non-small cell lung cancer, and is an important supplement to the detection of common EGFR mutations.

However, in the process of actually detecting rare mutations of the EGFR gene, there are still problems such as the quality of the detection samples. Although tumor tissue and tumor cytology samples are the best samples for mutation detection, such samples are difficult to obtain. Detection of free nucleic acid (Circulating free DNA, "cfDNA") in plasma, also known as "liquid biopsy", avoids the need for biopsy of tumor tissue, and is a very useful diagnostic application in clinical practice. The use of liquid biopsies offers the possibility of repeated blood sampling, allowing the tracking of cfDNA changes during tumorigenesis or during cancer treatment, thereby monitoring disease changes.

However, it is difficult to use cell-free nucleic acid (such as cfDNA) as a biomarker in tumor patients, especially the application of cfDNA detection to accurately and specifically detect gene mutations currently has huge technical challenges. First, the content of cfDNA in blood varies from person to person, and in most cases it is very low, and the quality and content of tumor-derived cell-free nucleic acid (Circulating tumor DNA, referred to as "ctDNA") is even more uneven.

Not only that, the specificity of cfDNA detection methods needs to be improved. Douillard et al. reported that the detection of EGFR mutations using plasma was only 65% concordant with tumor tissue detection.

In addition, although there are some methods based on cfDNA to detect rare gene mutations of EGFR, the sensitivity and specificity of these methods are still unsatisfactory.

Therefore, there is an urgent need in the art to develop a cfDNA-based method for detecting EGFR gene mutations with high sensitivity and high specificity.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for detecting EGFR gene mutation with high sensitivity and high specificity based on cfDNA.

In a first aspect of the present invention, there is provided a reagent for detecting gene mutation, the reagent is selected from the group consisting of:

(a) a first primer pair for detecting EGFR S768 mutation, wherein the first primer pair includes primers shown in SEQ ID Nos: 1 and 2;

(b) a second primer pair for detecting EGFR L861 mutation, wherein the second primer pair includes primers shown in SEQ ID Nos: 5 and 6;

(c) a third primer pair for detecting EGFR G719 mutation, wherein the third primer pair includes the primers shown in SEQ ID Nos: 9 and 10;

(d) A combination of the above (a), (b) and (c).

In another preferred embodiment, the sequence of the EGFR S768 is located in NG_007726.3:167254～167377.

In another preferred embodiment, the nucleic acid sequence of the wild-type sequence of EGFR S768 is shown in SEQ ID NO.:15.

In another preferred embodiment, the EGFR S768I mutation refers to the mutation of serine S at position 768 of the EGFR protein amino acid sequence to isoleucine I (ie, S768I).

In another preferred embodiment, the EGFR S768I mutation refers to the mutation of guanine G at position 2303 of the EGFR gene nucleic acid sequence to thymine T (EGFR c.2303G>T).

In another preferred example, the sequence of the EGFR L861 is located in NG_007726.3:177753-177832.

In another preferred example, the nucleic acid sequence of the wild-type sequence of EGFR L861 is shown in SEQ ID NO.:17.

In another preferred embodiment, the EGFR L861Q mutation refers to the mutation of leucine L at position 861 of the EGFR protein amino acid sequence to glutamine Q (ie, L861Q).

In another preferred embodiment, the EGFR L861Q mutation refers to the mutation of thymine T at position 2582 of the EGFR gene nucleic acid sequence to adenine A (EGFR c.2582T>A).

In another preferred embodiment, the sequence of the EGFR G719 is located in NG_007726.3:159920～160027.

In another preferred example, the nucleic acid sequence of the wild-type sequence of EGFR G719 is shown in SEQ ID NO.:19.

In another preferred embodiment, the EGFR G719 mutation refers to G719S, G719C, and G719A mutations.

In another preferred embodiment, the EGFR G719S mutation refers to the mutation of glycine G at position 719 of the EGFR protein amino acid sequence to serine S (ie, G719S).

In another preferred embodiment, the EGFR G719S mutation refers to the mutation of guanine G at position 2155 of the EGFR gene nucleic acid sequence to adenine A (EGFR c.2155G>A).

In another preferred embodiment, the EGFR G719C mutation refers to the mutation of glycine G at position 719 of the EGFR protein amino acid sequence to cysteine C (ie, G719C).

In another preferred embodiment, the EGFR G719C mutation refers to the mutation of guanine G at position 2155 of the EGFR gene nucleic acid sequence to thymine T (ie, 2155G>T).

In another preferred embodiment, the EGFR G719A mutation refers to the mutation of glycine G at position 719 of the EGFR protein amino acid sequence to alanine A (ie, G719A).

In another preferred embodiment, the EGFR G719A mutation refers to the mutation of guanine G at position 2156 of the EGFR gene nucleic acid sequence to cytosine C (EGFR c.2156G>C).

In another preferred embodiment, the reagent also includes:

(a1) the first probe used in combination with the first primer pair, wherein the first probe is selected from the group consisting of: the probe shown in SEQ ID No: 3, the probe shown in SEQ ID No: 4, or a combination thereof;

In another preferred embodiment, the reagent also includes:

(b1) a second probe used in conjunction with a second primer pair, wherein the second probe is selected from the group consisting of: the probe shown in SEQ ID No: 7, the probe shown in SEQ ID No: 8, or a combination thereof.

In another preferred embodiment, the reagent also includes:

(c1) a third probe used in conjunction with a third primer pair, wherein the third probe is selected from the group consisting of the probe shown in SEQ ID No: 11, the probe shown in SEQ ID No: 12, The probe set forth in SEQ ID No: 13, the probe set forth in SEQ ID No: 14, or a combination thereof.

In another preferred embodiment, the first probe, the second probe and the third probe are single-stranded nucleic acid probes.

In another preferred embodiment, the nucleic acid probe contains one or more locked nucleotides.

In another preferred embodiment, the structure (5'-3') of the first probe is shown in formula I:

Z1-Z2-Z3 I

in,

Z1 is a fluorescent group;

Z2 is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

Z3 is a quenching group;

"-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

In another preferred embodiment, the Z2-specific nucleic acid sequence targets the S768 site of wild-type EGFR.

In another preferred embodiment, the Z2-specific nucleic acid sequence targets the mutant EGFR S768I site.

In another preferred embodiment, the Z2 contains locked nucleotide modifications.

In another preferred embodiment, the sequence of described Z2 is selected from the following group:

TGGCCA+GCGTGGACA (SEQ ID No: 3);

TGGCC+A+T+CGTGGACA (SEQ ID No: 4);

In each formula, "+T" represents a locked nucleotide T, "+C" represents a locked nucleotide C, and "+G" represents a locked nucleotide G.

In another preferred embodiment, the fluorescent groups are independently located at the 5' end, the 3' end and the middle of the nucleic acid probe.

In another preferred embodiment, the fluorescent group and the quenching group are each independently located at the 5' end, the 3' end, and/or the middle.

In another preferred embodiment, the fluorescent group includes a fluorescent group cross-linked with the DNA probe.

In another preferred embodiment, the fluorescent group is selected from the group consisting of FAM, VIC, HEX, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Texas Red, or a combination thereof.

In another preferred embodiment, the quenching group is selected from the group consisting of DABCYL, TAMRA, BHQ1, BHQ2, BHQ3, MGB, BBQ-650, TQ1-TQ6, QSY 7carboxylic acid, TQ7, eclipse, or a combination thereof.

In another preferred embodiment, the nucleic acid probe is WTP-EGFR S768 (SEQ ID No: 3).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR S768I (SEQ ID No: 4).

In another preferred example, the structure (5'-3') of the second probe is shown in formula II:

Z1'-Z2'-Z3' II

in,

Z1' is a fluorescent group;

Z2' is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

Z3' is a quenching group;

"-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

In another preferred embodiment, the Z2'-specific nucleic acid sequence targets the L861 site of wild-type EGFR.

In another preferred embodiment, the Z2'-specific nucleic acid sequence targets the mutant EGFR L861Q site.

In another preferred embodiment, the Z2' contains a locked nucleotide modification.

In another preferred embodiment, the sequence of described Z2' is selected from the following group:

CAAA+C+T+GCTGGGTG (SEQ ID No: 7);

CAAA+C+A+GCTGGGTG (SEQ ID No: 8);

In each formula, "+T" represents a locked nucleotide T, "+C" represents a locked nucleotide C, and "+G" represents a locked nucleotide G.

In another preferred embodiment, the nucleic acid probe is WTP-EGFR L861 (SEQ ID No: 7).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR L861Q (SEQ ID No: 8).

In another preferred example, the structure (5'-3') of the third probe is shown in formula III:

Z1”-Z2”-Z3” III

in,

Z1" is a fluorescent group;

Z2'' is a specific complementary nucleic acid sequence that contains locked nucleotides or does not contain locked nucleotides;

Z3" is a quenching group;

"-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

In another preferred embodiment, the Z2" specific nucleic acid sequence targets the G719 site of wild-type EGFR.

In another preferred embodiment, the Z2" specific nucleic acid sequence targets a mutant EGFR site selected from the group consisting of G719S, G719C, G719A or a combination thereof.

In another preferred embodiment, the Z2" contains a locked nucleotide modification.

In another preferred embodiment, the sequence of said Z2" is selected from the following group:

TGCTGG+GCTCCGGT (SEQ ID No: 11);

TGCTG+A+GCTCCGGT (SEQ ID No: 12);

TGCTGG+CCTCCGGT (SEQ ID No: 13);

TGCTG+T+GCTCCGGT (SEQ ID No: 14);

In each formula, "+A" means locked nucleotide A, "+T" means locked nucleotide T, "+C" means locked nucleotide C, and "+G" means locked nucleotide G.

In another preferred embodiment, the nucleic acid probe is WTP-EGFR G719 (SEQ ID No: 11).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR G719S (SEQ ID No: 12).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR G719C (SEQ ID No: 13).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR G719A (SEQ ID No: 14).

In the second aspect of the present invention, a kit is provided, the kit contains the reagent for detecting gene mutation according to the first aspect.

In another preferred example, the kit further contains a first probe used in conjunction with the first primer pair; and/or a second probe used in conjunction with the second primer pair; and/or a third primer used in conjunction For use with the third probe.

In another preferred embodiment, the first primer pair, the second primer pair, the third primer pair, the first probe, the second probe, and the third probe are as described above.

In another preferred embodiment, the kit includes the first primer pair shown in SEQ ID Nos: 1 and 2, and the first probe shown in SEQ ID Nos: 3 and 4.

In another preferred embodiment, the kit includes the second primer pair shown in SEQ ID Nos: 5 and 6, and the second probe shown in SEQ ID Nos: 7 and 8.

In another preferred embodiment, the kit includes the third primer pair shown in SEQ ID Nos: 9 and 10, and the third probes shown in SEQ ID Nos: 11, 12, 13 and 14.

In the third aspect of the present invention, there is provided the use of the reagent for detecting gene mutation according to the first aspect or the use of the kit according to the second aspect, for preparing a diagnostic product, the diagnostic product using The effect of using EGFR-targeted drugs in pre-assessed subjects.

In another preferred embodiment, the EGFR-targeted drug is selected from the following group: such as cetuximab, panitumumab, gefitinib, erlotinib, afatinib, icotinib .

In another preferred embodiment, the product is for detection of cfDNA samples.

In another preferred embodiment, the cfDNA is from the blood, plasma, or serum of the subject.

In another preferred embodiment, the subject is a tumor patient.

In another preferred embodiment, the tumor is an EGFR-positive tumor.

In another preferred embodiment, the tumor is selected from the group consisting of colon cancer, liver cancer, lung cancer, gastric cancer, breast cancer, or a combination thereof.

In a fourth aspect of the present invention, a method for detecting whether a sample to be tested contains a gene mutation is provided, comprising the steps of:

(S1) a PCR reaction system is provided, the PCR reaction system contains a sample to be tested as a template and a primer pair for amplification, and the primer pair is selected from the following group:

(a) a first primer pair for detecting EGFR S768 mutation, wherein the first primer pair includes primers shown in SEQ ID Nos: 1 and 2;

(b) a second primer pair for detecting EGFR L861 mutation, wherein the second primer pair includes primers shown in SEQ ID Nos: 5 and 6;

(c) a third primer pair for detecting EGFR G719 mutation, wherein the third primer pair includes the primers shown in SEQ ID Nos: 9 and 10;

(d) a combination of (a), (b) and (c) above;

The reagent for detecting gene mutation described in the first aspect;

(S2) PCR reaction is performed on the PCR reaction system of step (S1), thereby obtaining an amplification product;

(S3) Analyzing the amplification product generated in step (S2) to obtain an analysis result of whether the sample to be tested contains a gene mutation.

In another preferred embodiment, the analysis result is a qualitative result.

In another preferred embodiment, the gene mutation is selected from: S768I, L861Q, G719X (including G719S, G719C, G719A) or a combination thereof.

In another preferred embodiment, the PCR reaction system is a digital PCR reaction system.

In another preferred embodiment, the digital PCR is ddPCR.

In another preferred example, the concentration of the target nucleic acid molecule to be detected in the ddPCR droplet is 1 to 1×10 ¹⁵ copies/ml, preferably 1 to 10 ¹⁰ copies/ml, more preferably 1 to 10 ⁵ copies/ml.

In another preferred embodiment, in step (S2), the annealing temperature of the PCR reaction for S768I gene mutation detection is 60±2°C, preferably 60±1°C, more preferably 60±0.5°C.

In another preferred embodiment, in step (S2), the annealing temperature of the PCR reaction for L861Q gene mutation detection is 56±2°C, preferably 56±1°C, more preferably 56±0.5°C.

In another preferred embodiment, in step (S2), the annealing temperature of the PCR reaction for G719X gene mutation detection is 58±2°C, preferably 58±1°C, more preferably 58±0.5°C.

In another preferred example, the PCR reaction system also contains:

(a1) the first probe used in combination with the first primer pair, wherein the first probe is selected from the group consisting of: the probe shown in SEQ ID No: 3, the probe shown in SEQ ID No: 4, or a combination thereof; and/or

(b1) a second probe used in conjunction with a second primer pair, wherein the second probe is selected from the group consisting of: the probe shown in SEQ ID No: 7, the probe shown in SEQ ID No: 8, or a combination thereof.

(c1) a third probe used in conjunction with a third primer pair, wherein the third probe is selected from the group consisting of the probe shown in SEQ ID No: 11, the probe shown in SEQ ID No: 12, The probe set forth in SEQ ID No: 13, the probe set forth in SEQ ID No: 14, or a combination thereof.

In another preferred example, the probes (SEQ ID No: 3, SEQ ID No: 7 and SEQ ID No: 11) used to detect wild-type genes use the same first fluorescent label (eg HEX, FAM).

In another preferred example, the probes (SEQ ID Nos: 4, 8, 12, 13 and 14) used to detect mutant genes use the same second fluorescent label (such as HEX, FAM), and the first fluorescent The label and the second fluorescent label are different.

In another preferred embodiment, the probe contains locked nucleotides.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the method is an in vitro method.

In another preferred embodiment, the detection accuracy of the method is 0.06%-1%, preferably 0.0625%-0.08%.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the results of EGFR S768 5-pair primer PCR electrophoresis (M: DNA Marker).

Figure 2 shows the results of EGFR L861 5-pair primer PCR electrophoresis (M: DNA Marker).

Figure 3 shows the results of EGFR G719 5-pair primer PCR electrophoresis (M: DNA Marker).

Figure 4 shows the optimization of the annealing temperature of the Taqman-ddPCR method for the detection of the EGFR gene S768I mutation. Wells C01, C06, and C12 represent annealing temperature gradients of 60°C, 58°C, and 56°C. Where A is the 1-D amplitude map of the positive control (100% S768I mutation) detected by the FAM channel; B is the 1-D amplitude map of the negative control detected by the HEX channel

Figure 5 shows the optimization of the annealing temperature of the Taqman-ddPCR method for the detection of EGFR gene L861Q mutation. Wells A-H represent annealing temperature gradients of 65°C, 64.5°C, 63.3°C, 61.4°C, 59°C, 57°C, 56°C, 55°C. Wherein A is the 1-D amplitude map of the positive control (100% L861Q mutation) detected by the FAM channel; B is the 1-D amplitude map of the negative control detected by the HEX channel.

Figure 6 shows the optimization of the annealing temperature of the Taqman-ddPCR method for the detection of the EGFR gene G719X mutation. Wells F10, F06, and F02 represent annealing temperature gradients of 58°C, 56°C, and 54°C. Where A is the 1-D amplitude map of the positive control (100% G719S/A/C mutation) detected by the FAM channel; B is the 1-D amplitude map of the negative control detected by the HEX channel

Figure 7 shows the validation of the detection concentration for the EGFR S768I gene mutation. It can be seen from the figure that the linearity of this detection method is very good in all detection concentrations (0.06%-1%), that is, the sensitivity of this method reaches 0.06%

Figure 8 shows the validation of the detection concentration for the EGFR L861Q gene mutation. It can be seen from the figure that the linearity of this detection method is very good in all detection concentrations (0.06%-1%), that is, the sensitivity of this method reaches 0.06%.

Figure 9 shows the validation of the detection concentration for the EGFR G719X gene mutation. It can be seen from the figure that the linearity of this detection method is very good in all detection concentrations (0.06%-1%), that is, the sensitivity of this method reaches 0.06%.

Figure 10 shows that case "HP159" was negative for EGFR by digital PCR. From top to bottom are the detection 2D map of S768I, L861Q, G719X (2D amplitude map of FAM-HEX channel). where green is the wild-type signal.

Figure 11 shows a two-dimensional map of case "HP186" detected by digital PCR for EGFR S768I (2D map of the FAM-HEX channel). Among them, blue and orange are mutation signals, indicating that the case "HP186" was positive for EGFR S768I by digital PCR.

Figure 12 shows a two-dimensional map (2D map of the FAM-HEX channel) for the detection of EGFR L861Q by digital PCR in case "BH0139". Among them, blue and orange are mutation signals, indicating that they are positive for EGFR L861Q detected by digital PCR.

Figure 13 shows a two-dimensional map (2D map of the FAM-HEX channel) for the detection of EGFR G719S by digital PCR in case "MS1025". Among them, blue and orange are mutation signals, indicating that they are positive for EGFR G719S detected by digital PCR.

Figure 14 shows that case "MS3248" was positive for EGFR G719C by digital PCR. 2D map of the FAM-HEX channel; blue and orange are mutant signals.

Figure 15 shows a 2D plot (2D plot of FAM-HEX channel) for the detection of EGFR G719A by digital PCR in case "MS2006". Among them, blue and orange are mutation signals, indicating that they are positive for EGFR G719A detected by digital PCR.

Figure 16 shows a two-dimensional plot (2D plot of FAM-HEX channel) for the detection of EGFR G719X by digital PCR in case "MS2006". Among them, blue and orange are mutation signals, indicating that they are positive for EGFR G719X detected by digital PCR.

Detailed ways

After extensive and in-depth research, the inventors can effectively improve the effect of gene mutation detection through a large number of screenings, especially by optimizing the optimization of primer sequences and probes, combined with the digital PCR platform, and break through the existing gene mutation detection technology. Pathological tissue as a starting material has problems such as low accuracy and low sensitivity, and provides a method for detecting EGFR gene mutation with high specificity, high sensitivity and strong anti-interference ability. On this basis, the present inventors have completed the present invention.

Specifically, the present invention provides a detection method for EGFR S768I, EGFR L861Q, EGFR G719S, G719C, G719A and mutated genes, that is, by providing three pairs of specially optimized EGFR S768, EGFR L861, and EGFR G719. Primer pairs and specially optimized probes for EGFR S768I, EGFR L861Q, EGFR G719S, G719C, and G719A mutant genes were used to establish a digital PCR detection system to qualitatively and quantitatively detect the mutation of EGFR genes. When the method and reagent of the present invention are used to detect EGFR gene mutation, it has unexpectedly high sensitivity and high specificity, and can detect samples of different difficulty.

the term

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Additional definitions are set forth throughout the application.

The term "about" may refer to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of," or "consisting of."

Sequence identity is obtained by comparing two aligned sequence, and determine the number of positions where identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

Digital PCR (digital PCR) technology

Digital PCR (digital PCR) technology, based on single-molecule PCR method for counting nucleic acid quantification, is an absolute quantitative method. It mainly adopts the microfluidic or microdroplet method in the current hot research field of analytical chemistry to disperse a large amount of diluted nucleic acid solution into the microreactors or microdroplets of the chip, and the number of nucleic acid templates in each reactor is less than or equal to 1 indivual. In this way, after the PCR cycle, the fluorescence signal of each droplet is analyzed after the amplification, the reactor with the nucleic acid molecule template will give a fluorescence signal, and the reactor without the template will have no fluorescence signal. Based on the relative proportions and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated.

Compared with conventional qPCR, digital PCR can accurately quantitatively analyze and detect target nucleic acid molecules with high sensitivity. The method of analyzing the results of conventional qPCR is an analog method, wherein the digital PCR method, the results of which are analyzed by digital methods (since the resulting signal has a value of "0" or "1"), has the ability to analyze large volume samples. , The advantages of simultaneous detection of different samples and different tests at the same time. Digital PCR is a technique that allows absolute quantification of DNA samples using single-molecule counting without a standard curve, and allows for more precise absolute quantification of individual droplets per well by PCR (see Gudrun Pohl and le-Ming Shih). , Principle and applications of digital PCR (Principle and applications of digital PCR), Expert Rev. Mol. Diagn. 4(1), 41-47 (2004)). Digital PCR has the advantages of high sensitivity, accurate quantification without standard curve, and simple operation.

In digital PCR, each droplet containing the sample gene template, amplification primers and fluorescent probes prepared for dilution to an average copy number of 0.5-1 is dispensed into a single well and microemulsion PCR is performed. Wells showing a fluorescent signal are then counted as a value of "1" because samples with a gene copy number of 1 were assigned to the wells and showed a fluorescent signal after amplification, while wells that did not show a signal were counted as "0" because Samples with a gene copy number of 0 were dispensed into the wells and showed no fluorescent signal due to no amplification. In this way, absolute quantification can be achieved.

primer

Primer refers to a macromolecule with a specific nucleotide sequence that stimulates synthesis at the initiation of nucleotide polymerization, and is linked to the reactant in the form of covalent bonds. The primers are usually two synthetic oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to another DNA template strand at the other end of the target region.

In the present invention, in order to improve the sensitivity of the detection system, the corresponding gene fragments in the detection system are pre-amplified, so that primers corresponding to the sequences where the mutation is located are designed.

In a preferred example, according to the sequence of EGFR S768, multiple pairs of primers are designed at the upstream and downstream of the EGFR gene S768 site respectively. After experimental testing, the optimal primer pair is finally determined to be SEQ ID Nos: 1 and 2.

In another preferred example, according to the sequence of EGFR L861, multiple pairs of primers were designed on the upstream and downstream of the EGFR gene L861 site respectively. After experimental tests, the optimal primer pairs were finally determined as SEQ ID Nos: 5 and 6.

In another preferred example, according to the sequence of EGFR G719, multiple pairs of primers were designed at the upstream and downstream of the EGFR gene G719 site respectively. After experimental tests, the optimal primer pairs were finally determined to be SEQ ID Nos: 9 and 10.

probe

In this context, "probe", "nucleic acid probe" and "gene probe" can be replaced, and refer to a nucleic acid sequence (DNA or RNA) with a detectable label and a known sequence that is complementary to the target gene. The gene probe is combined with the target gene through molecular hybridization to generate a hybridization signal, which can display the target gene from the vast genome. According to the hybridization principle, the nucleic acid sequence used as a probe must meet at least the following two conditions: ① It should be single-stranded, if it is double-stranded, it must be denatured first; ② It should have a label that can be easily detected. Nucleic acid probes can include the entire gene or only a part of the gene; it can be DNA itself or RNA transcribed from it. In the present invention, the probe also refers to a modified primer, and the modified primer has chemical modification groups at both ends or in the middle. These chemical modifications have special functions including but not limited to: signal indication, enhancement and reaction connection of substances, etc.

The structure (5'-3') of the first probe of the present invention is shown in formula I:

Z1-Z2-Z3 I

in,

Z1 is a fluorescent group;

Z2 is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

Z3 is a quenching group;

"-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

In another preferred embodiment, the Z2-specific nucleic acid sequence targets the S768 site of wild-type EGFR.

In another preferred embodiment, the Z2-specific nucleic acid sequence targets the mutant EGFR S768I site.

In another preferred embodiment, the Z2 contains locked nucleotide modifications.

In another preferred embodiment, the sequence of described Z2 is selected from the following group:

TGGCCA+GCGTGGACA (SEQ ID No: 3);

TGGCC+A+T+CGTGGACA (SEQ ID No: 4);

In each formula, "+T" represents a locked nucleotide T, "+C" represents a locked nucleotide C, and "+G" represents a locked nucleotide G.

In another preferred embodiment, the fluorescent groups are independently located at the 5' end, the 3' end and the middle of the nucleic acid probe.

In another preferred embodiment, the fluorescent group and the quenching group are each independently located at the 5' end, the 3' end, and/or the middle.

In another preferred embodiment, the fluorescent group includes a fluorescent group cross-linked with the DNA probe.

In another preferred embodiment, the fluorescent group is selected from the group consisting of FAM, VIC, HEX, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Texas Red, or a combination thereof.

In another preferred embodiment, the quenching group is selected from the group consisting of DABCYL, TAMRA, BHQ1, BHQ2, BHQ3, MGB, BBQ-650, TQ1-TQ6, QSY 7carboxylic acid, TQ7, eclipse, or a combination thereof.

In another preferred embodiment, the nucleic acid probe is WTP-EGFR S768 (SEQ ID No: 3).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR S768I (SEQ ID No: 4).

The structure (5'-3') of the second probe of the present invention is shown in formula II:

Z1'-Z2'-Z3' II

in,

Z1' is a fluorescent group;

Z2' is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

Z3' is a quenching group;

"-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

In another preferred embodiment, the Z2'-specific nucleic acid sequence targets the L861 site of wild-type EGFR.

In another preferred embodiment, the Z2'-specific nucleic acid sequence targets the mutant EGFR L861Q site.

In another preferred embodiment, the Z2' contains a locked nucleotide modification.

In another preferred embodiment, the sequence of described Z2' is selected from the following group:

CAAA+C+T+GCTGGGTG (SEQ ID No: 7);

CAAA+C+A+GCTGGGTG (SEQ ID No: 8);

In each formula, "+T" represents a locked nucleotide T, "+C" represents a locked nucleotide C, and "+G" represents a locked nucleotide G.

In another preferred embodiment, the nucleic acid probe is WTP-EGFR L861 (SEQ ID No: 7).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR L861Q (SEQ ID No: 8).

The structure (5'-3') of the third probe of the present invention is shown in formula III:

Z1”-Z2”-Z3” III

in,

Z1" is a fluorescent group;

Z2'' is a specific complementary nucleic acid sequence that contains locked nucleotides or does not contain locked nucleotides;

Z3" is a quenching group;

"-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

In another preferred embodiment, the Z2" specific nucleic acid sequence targets the G719 site of wild-type EGFR.

In another preferred embodiment, the Z2" specific nucleic acid sequence targets a mutant EGFR site selected from the group consisting of G719S, G719C, G719A or a combination thereof.

In another preferred embodiment, the Z2" contains a locked nucleotide modification.

In another preferred embodiment, the sequence of said Z2" is selected from the following group:

TGCTGG+GCTCCGGT (SEQ ID No: 11);

TGCTG+A+GCTCCGGT (SEQ ID No: 12);

TGCTGG+CCTCCGGT (SEQ ID No: 13);

TGCTG+T+GCTCCGGT (SEQ ID No: 14);

In each formula, "+A" means locked nucleotide A, "+T" means locked nucleotide T, "+C" means locked nucleotide C, and "+G" means locked nucleotide G.

In another preferred embodiment, the nucleic acid probe is WTP-EGFR G719 (SEQ ID No: 11).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR G719S (SEQ ID No: 12).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR G719C (SEQ ID No: 13).

In another preferred embodiment, the nucleic acid probe is MTP-EGFR G719A (SEQ ID No: 14).

Modification of primers and probes

In the present invention, the nucleic acid sequences of the primers include unmodified or modified primer sequences.

Preferably, the inventors can significantly improve the specificity of the probe by modifying the probe with locked nucleotides, thereby improving the sensitivity and specificity of the detection result.

In a preferred embodiment of the present invention, the modification is selected from: Phosphorylation, Biotin, Digoxigenin, internal amino modification, 5' amino modification, 3' amino modification, sulfhydryl group (Thiol), Spacer, Phosphorthioate, DeoxyUridine (dU), DeoxyInosine (dI), or a combination thereof.

Phosphorylation modification: 5' phosphorylation can be used for linkers, cloning and gene construction, and ligase-catalyzed ligation reactions. 3' phosphorylation is resistant to 3' exonuclease digestion in related experiments, and is also used to prevent DNA polymerase-catalyzed DNA chain extension reactions.

Biotin modification: The primers are labeled with biotin, which can be used for non-radioactive immunoassays to detect proteins, intracellular chemical staining, cell isolation, nucleic acid isolation, hybridization to detect specific DNA/RNA sequences, ion channel conformational changes, etc.

Modification of Digaoxin: Digaoxin is linked to the C5 position of uracil through an 11-atom spacer arm, and the hybridized Digaoxin probe can be detected by anti-Digaoxin antibody. Digao-labeled probes can be used in various hybridization reactions, such as DNA-DNA hybridization (Southern blotting), DNA-RNA hybridization (Northern blotting), dot blotting (Dotblotting), clone hybridization, in situ hybridization and enzyme-linked immunoassay ( ELISA).

Internal amino modification: C6-dT aminolinker is mainly used to add to thymine residues for internal modification. The modified amino group is 10 atoms away from the main chain, which can be used for further labeling and enzymatic ligation (such as alkaline phosphatase), and dT-Dabcyl, dT-Biotin, and dT-Digoxingenin modifications mediated by internal amino modifications are currently available.

5' Amino Modification: It can be used to prepare functionalized oligonucleotides, which are widely used in DNA chips (DNA Microarray) and multiple labeling diagnostic systems. At present, there are two kinds of 5'C6 amino modification and 5'C12 amino modification. The former can be used to connect some compounds that will not affect their function even if they are close to the oligonucleotide, and the latter is used for the connection of affinity purification groups and some fluorescence labeling, especially when fluorescence may be quenched by the label being too close to the DNA strand.

3' Amino Modifications: 3'C6 Amino Modifications are currently available. It can be used to design new diagnostic probes and antisense nucleotides, for example, the 5' end can be labeled with highly sensitive 32P or fluorescein while the 3' can be modified with amino groups for other linkages. In addition, 3' modification can inhibit 3' exonuclease digestion, which can be used in antisense experiments.

Sulfhydryl Modifications: 5'-Sulfhydryl groups are similar in many ways to amino modifications. Thiol groups can be used to attach various modifications such as fluorescent labels and biotin. For example, thiol-linked fluorescent probes can be prepared in the presence of iodoacetic acid and maleimide derivatives. The sulfhydryl modification of 5' mainly uses 5' sulfhydryl to modify the monomer (5'-Thiol-Modifier C6-CE Phosphoramidite or Thiol-Modifier C6 S-S CE Phosphoramidite). After modification with 5'-Thiol-Modifier C6-CE monomer, silver nitrate must be oxidized to remove the protective group (trityl), while Thiol-Modifier C6 S-S CE monomer must be modified with DTT to reduce the disulfide bond to sulfhydryl.

Spacer modification: Spacer can provide the necessary space for oligonucleotide labeling to reduce the interaction between the labeling group and oligonucleotide. It is mainly used in the study of DNA hairpin structure and double-stranded structure. The C3 spacer is primarily used to mimic the three-carbon spacer between the 3' and 5' hydroxyls of ribose, or to "replace" an unknown base in a sequence. 3'-Spacer C3 is used to introduce a 3' spacer to prevent the 3' exonuclease and 3' polymerase from functioning. Spacer 18 is often used to introduce a strongly hydrophilic group.

Thio-modified: Thio-modified oligonucleotides are mainly used in antisense experiments to prevent degradation by nucleases. You can choose all-thio, but the Tm value of the oligonucleotide will decrease with the increase of thio bases. To reduce this effect, 2-5 bases at both ends of the primer can be thio-modified , usually 3 bases at 5' and 3' can be selected for sulfur modification.

Deoxyuracil modification: Deoxyuracil can be inserted into oligonucleotides to increase the melting point temperature of the duplex to increase the stability of the duplex. The replacement of each deoxythymine by deoxyuracil can increase the double-stranded melting point temperature of 1.7°C.

Deoxyhypoxanthine modification: Deoxyhypoxanthine is a naturally occurring base. Although it is not a universal base in the true sense, it is relatively more stable than other base mismatches when combined with other bases. The binding ability of deoxyhypoxanthine to other bases is dI:dC>dI:dA>dI:dG>dI:dT. Under the catalysis of DNA polymerase, deoxyhypoxanthine is firstly combined with dC.

cfDNA

Circulating free DNA ("cfDNA") in plasma, also known as "liquid biopsy", avoids the need for biopsy of tumor tissue and is a very useful diagnostic application in clinical practice. The use of liquid biopsies offers the possibility of repeated blood sampling, allowing the tracking of cfDNA changes during tumorigenesis or during cancer treatment to monitor disease changes (Cell-free nucleic acids as biomarkers in cancer patients). However, the application of cfDNA detection to accurately and specifically detect gene mutations currently has huge technical challenges. First of all, the content of cfDNA in blood varies from person to person, and in most cases it is very low, and the quality and content of tumor-derived cell-free nucleic acid (Circulating tumorDNA, referred to as "ctDNA") is even more uneven. Not only that, the specificity of cfDNA detection methods needs to be improved. Douillard et al reported that the concordance between the detection of EGFR mutations in plasma and tumor tissue detection results was only 65%.

The main advantages of the present invention include:

1. High sensitivity: Since this method adopts a digital PCR platform, the reaction system can be divided into about 20,000 micro-reactions, and a single copy mutation can theoretically be detected, which has the advantage of sensitivity unmatched by other technologies. The detection method of the present invention can reach the lowest detection limit of 0.06% through verification.

2. Strong specificity: The designed specific primers are respectively aimed at the sequences of EGFR gene S768, EGFR gene L861, and EGFR gene G719, which can specifically amplify the wild-type and mutant templates of the target position; the designed specific probe covers Mutation sites, designed for wild-type (3 in total) and mutant (5 in total) of EGFR S768, L861, and G719. The 5' end of the wild-type probe is modified with a HEX fluorophore, and the 5' of the mutant probe At the same time, the EGFR probe has a locked nucleic acid (LNA) modification at the mutated base, which greatly enhances the binding force of the nucleotide here. The EGFR probe has BHQ1 at the 3' end, which can The design of the above-mentioned primers and probes greatly improves the specificity of detection by effectively distinguishing templates with a base difference.

3. The requirements for the type and quality of the samples are loose, and the anti-interference ability is strong. Due to its high sensitivity, the applicable sample types of the present invention are not only fresh tissue samples and paraffin sections commonly used in general methods, but also peripheral blood samples (this sample is easier to obtain, but the DNA content is low and fragmented); The uniqueness of its digital PCR platform is that the reaction system can be divided into about 20,000 small systems, and the interfering substances can also be divided into about 20,000 parts, which can greatly reduce the impact of interfering substances on the reaction, and of course can detect more complex backgrounds. sample. This is something no other platform can do.

4. The positive interpretation method is simple: since the present invention adopts the absolute quantitative method, it is not necessary to set a reference standard curve, and the result can be determined whether it contains the target mutation template according to the two-dimensional graph of fluorescence (Table 1).

Table 1. Test result table

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989), or according to the conditions suggested by the manufacturer . Percentages and parts are weight percentages and parts unless otherwise specified.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

Sequences of primers, probes and amplified fragments

In the embodiment, the nucleic acid sequence information of the primers, probes and amplified DNA fragments used are shown in Table 2:

Table 2: Nucleic acid sequences of primers, probes and DNA fragments

Note: "+A" means locked nucleotide A, "+T" means locked nucleotide T, "+C" means locked nucleotide C, "+G" means locked nucleotide G.

Example 1 Screening of primer pairs

According to the EGFR S768 site, 5 pairs of primers were designed (primers were synthesized from Shanghai Sangon Biological Co., Ltd.), and the specific sequences were shown in Table 3 below.

Table 3. Screening of 5 primer pairs for amplification of EGFR S768

According to the EGFR L861 site, 5 pairs of primers were designed (primers were synthesized from Shanghai Sangon Biological Co., Ltd.), and the specific sequences were shown in Table 4 below.

Table 4. Screening of 5 primer pairs for amplification of EGFR L861

According to the EGFR G719 site sequence, 5 pairs of primers were designed (the primers were synthesized from Shanghai Sangon Biological Co., Ltd.), and the specific sequences are shown in Table 5:

Table 5. Screening of 5 primer pairs for amplification of EGFR G719

Screening PCR system: 0.2ul of Taq HS polymerase, 2ul of 10×HS PCR buffer, 1.6ul of dNTP mixture, 1ul of upstream primer, 1ul of downstream primer, 5ul of tgDNA (human leukocyte genomic DNA, as template), and water to make up the system to 20ul .

Screening PCR program: 95°C for 10 min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 20s).

After PCR screening, S768 primer pair 2 (S768-F2/S768-R2), L861 primer pair 5 (L861-F5/L861-R5) and G719 primer pair 1 (G719-F1/G719-R1) amplified the target fragment The efficiency is high (bright band), the amplification product is short, and no primer-dimer is produced. The overall effect is the best, and it can be used as a primer pair for EGFR gene mutation detection. See Figures 1 to 3.

Example 2 Optimization of Taqman probe and digital PCR reaction program

In this example, Taqman probes were synthesized and optimized, and the digital PCR reaction program was optimized based on the optimized Taqman probes.

2.1 Taqman probe

In this example, the detected EGFR gene mutations are nucleotide mutations corresponding to S768I, L861Q, G719S, G719C, and G719A (Table 6);

Table 6: EGFR gene mutation information table

Detection site type of mutation COSMIC number genetic changes S768I point mutation COSM6241 c.2303G>T L861Q point mutation COSM6213 c.2582T>A G719S point mutation COSM6252 c.2155G>A G719C point mutation COSM6253 c.2155G>T G719A point mutation COSM6239 c.2156G>C

Based on the genome sequence and mutation sites of human EGFR, the corresponding Taqman probes were designed and optimized. The optimized Taqman probes contained specific locked nucleotides (+N), namely WTP-S768, MTP-S768I, WTP -L861, MTP-L861Q, WTP-G719, MTP-G719S, MTP-G719A, MTP-G719C, the specific information of the probe is shown in Table 2.

2.2 Optimization of digital PCR reaction program

Based on the optimal primer pairs determined in Example 1 (SEQ ID Nos: 1 and 2; SEQ ID Nos: 5 and 6; SEQ ID Nos: 9 and 10) and the optimized probes determined in Example 2.1, for digital PCR reactions The program is further optimized. The digital PCR reaction program optimization experiment needs to comprehensively analyze the digital PCR amplification of wild-type template and mutant template with the change of annealing temperature. The optimal annealing temperature should satisfy two conditions: the negative control (wild-type template) has a clean background (no droplets in the FAM channel), and the positive and negative signals in the mutant template are easy to distinguish (high fluorescence intensity in the FAM and HEX channels).

The experimental method is as follows:

S768I and L861Q use a single-plex detection system. The preparation is as follows: 2×ddPCR Supermix for Probe (NodUTP) 11ul, primer 1 (F) 1.1ul, primer 2 (R) 1.1ul, probe 1 (WTP) 0.55ul, probe 2 (MTP) 0.55ul, template 5.5ul, add water to make up to 22ul. (F stands for upstream primer, R stands for downstream primer, WTP stands for wild-type probe, MTP stands for mutant probe, and the corresponding primer probe is added according to the detected target site)

G719X adopts triple detection system, the preparation is as follows: 2×ddPCR Supermix for Probe(No dUTP) 11ul, Primer 1(G719-F) 1.1ul, Primer 2(G719-R) 1.1ul, Probe 1(WTP-G719) 0.55 ul, probe 2 (MTP-G719S) 0.55ul, probe 3 (MTP-G719C) 0.55ul, probe 4 (MTP-G719A) 0.55ul

Add templates in the sample preparation area in the following order: negative control, positive control. The negative control was normal human leukocyte genome (tgDNA), the positive controls were a solution containing 1% S768I mutant genomic DNA (the plasmid containing the S768I mutant sequence was transfected into 293T cells, and the genomic DNA was extracted and incorporated into tgDNA), containing 1% L861Q mutant genomic DNA solution (transfect the plasmid containing L861Q mutant sequence into 293T cells, extract genomic DNA, and incorporate tgDNA), 1% G719X mutant genomic DNA solution (containing G719S, containing G719C, containing G719A mutations) Sequenced genomic DNA, the content of each of the three mutations is 1%, this control can be used as a control for a single mutation detection of G719X, or as a control for multiple detection). Select a positive control according to the content of the assay.

In the droplet generation area, droplet generation is performed according to the requirements of the instrument.

PCR is performed in the sample analysis area.

S768I PCR program: 95°C for 10 min, 40 cycles {94°C for 30s, annealing temperature gradient (60°C, 58°C, 56°C) for 15s, 72°C for 15s}, 98°C for 10 min, ramp rate 2°C/s.

L861Q PCR program 95°C for 10min, 40 cycles {94°C for 30s, annealing temperature gradient (65°C, 64.5°C, 63.3°C, 61.4°C, 59°C, 57°C, 56°C, 55°C) for 15s, 72°C for 15s}, 98°C for 10min, the heating and cooling rate is 2°C/s.

G719X PCR program: 95°C for 10min, 40 cycles {94°C for 30s, annealing temperature gradient (58°C, 56°C, 54°C) for 15s, 72°C for 15s}, 98°C for 10min, ramp rate 2°C/s.

After completing the PCR, set according to the instrument and experimental requirements, select the FAM/HEX detection channel, and start reading the plate.

The results are shown in Figures 4 to 6. Wherein, the FAM channel indicates that the MTP probe detects the droplets of the mutant template and their fluorescence intensity (Amplitude), and the HEX channel indicates that the WTP probe detects the droplets of the wild-type template and its fluorescence intensity (Amplitude). FAM-HEX double-positive droplets represent the presence of both wild-type and mutant templates. The number of droplets in the FAM channel can infer whether there is a mutant template in the digital PCR system and can accurately calculate the copy number (copies/ul).

The results shown in Figures 4 and 6 show that S768I was annealed at 60°C for 15s, L861Q was annealed at 56°C for 15s, and G719X58°C was annealed for 15s. The digital PCR negative control was free of contamination, and the distinction between negative and positive signals was the most obvious, and the overall effect was better. optimal annealing temperature.

Example 3 Sensitivity verification of detection of EGFR gene mutation by digital PCR method

The wild-type template copy number (copies/ul), mutant template genome copy number (copies/ul) and mutation rate (%) were calculated according to Example 1, and both were diluted to 4000 copies/ul with TE. The tgDNA was incorporated into the mutant-containing genome template, so that the proportion of mutant template was 0.06%, 0.13%, 0.25%, 0.5%, and 1%, respectively, to prepare a gradient dilution template.

S768I and L861Q use a single-plex detection system. The preparation is as follows: 2×ddPCR Supermix for Probe (NodUTP) 11ul, primer 1 (F) 1.1ul, primer 2 (R) 1.1ul, probe 1 (WTP) 0.55ul, probe 2 (MTP) 0.55ul, template 5.5ul, add water to make up to 22ul. (F stands for upstream primer, R stands for downstream primer, WTP stands for wild-type probe, MTP stands for mutant probe, and the corresponding primer probe is added according to the detected target site)

G719X adopts triple detection system, the preparation is as follows: 2×ddPCR Supermix for Probe(No dUTP) 11ul, Primer 1(G719-F) 1.1ul, Primer 2(G719-R) 1.1ul, Probe 1(WTP-G719) 0.55 ul, probe 2 (MTP-G719S) 0.55ul, probe 3 (MTP-G719C) 0.55ul, probe 4 (MTP-G719A) 0.55ul

Add templates in the following order in the sample preparation area: blank control, negative control, serial dilution template. The blank control is water, the negative control is tgDNA, and the gradient dilution template is the wild-type template incorporating 0.06%-1% mutant template.

The PCR reaction system was formed into droplets according to the same method as in Example 1. PCR was performed according to the optimized PCR program: 95°C for 10 min, 40 cycles (94°C for 30s, 60°C/56°C/58°C for 15s, 72°C for 15s), 98°C for 10min, and the ramp rate was 2°C/s. Start the plate reading according to the instrument requirements.

The digital PCR results are shown in Figures 7 to 9. The digital PCR method of the present invention uses water or tgDNA as a template, and has clean background and no pollution. In addition, the digital PCR method of the present invention incorporates 0.06%-1% mutant template into the wild-type template, detects that the copy number of the wild-type template is normal, and detects that the proportion of mutant templates (mutant template divided by total template multiplied by 100%) and The theoretical ratios were consistent and linearly correlated with R2 values greater ^than 0.98. That is, the detection value of the digital PCR method of the present invention is accurate within the sensitivity range of 0.06%-1%, that is, the minimum detection sensitivity of the digital PCR method of the present invention is 0.06%.

Example 4 Detection of EGFR mutation in patients by digital PCR method to guide tumor-targeted drug use

The cases "HP159", "HP186", "BH0139", "MS1025", "MS3248", "MS2006", and "MS1039" were all patients with stage IV lung adenocarcinoma. The venous blood was collected, the plasma was centrifuged twice, and the plasma free nucleic acid was extracted by a free nucleic acid extraction kit. The extracted nucleic acid was quantified by Qubit. Store in a -20°C refrigerator in the sample preparation room after quantification.

See Example 2 for the digital PCR detection system of the present invention.

Templates were added in the following order in the sample preparation area: blank control, negative control, case-free nucleic acid, and positive control (1%).

The PCR reaction system was formed into droplets according to the same method as in Example 1. PCR was performed according to the optimized PCR program: 95°C for 10 min, 40 cycles (94°C for 30s, 60°C/56°C/58°C for 15s, 72°C for 15s), 98°C for 10min, and the ramp rate was 2°C/s. Turn on the instrument, set up as required, and start reading the plate. In this EGFR detection, the background of blank control and negative control was clean, and the copy number and mutation ratio of positive control were normal.

The nucleic acid sample test results of the cases are shown in Table 7 below.

HP159 was interpreted as negative (see Figure 10), and it did not contain S768I mutation, L861Q mutation and G719X mutation, and other gene mutations could be detected in the follow-up, and then medication guidance could be provided.

HP186 was interpreted as S768I positive (see Figure 11; the mutation rate was 23.5%), and targeted drug therapy such as afatinib is recommended;

Case BH0139 was interpreted as L861Q positive (see Figure 12; mutation rate was 1.8%), and targeted drug therapy such as afatinib is recommended;

Case MS1025 was interpreted as G719S positive (see Figure 13; mutation rate was 0.21%), and targeted drug therapy such as afatinib is recommended;

Case MS3248 was interpreted as G719C positive (see Figure 14; mutation rate was 1.6%), and targeted drug therapy such as afatinib is recommended;

Case MS2006 was interpreted as G719A positive (see Figure 15; mutation rate was 0.14%), and targeted drug therapy such as afatinib is recommended;

Case MS1039 was interpreted as G719X positive (see Figure 16; mutation rate was 2.3%), and targeted drug therapy such as afatinib therapy is recommended.

The specific test data of the above cases are shown in Table 7 below:

Table 7: Gene mutation results of case detection

discuss:

At present, the methods for detecting gene mutations mainly include:

(1) High resolution melting curve (HRM). HRM is a gene analysis technique that forms different morphological melting curves based on the different melting temperatures of single nucleotides. The detection sensitivity is around 1-10%. However, due to the high false positive rate of the dye method, sequencing verification is required in the later stage, resulting in a longer detection cycle.

(2) Probe amplification block mutation method (ARMS-qPCR). Using the principle that the 3'-terminal base of the PCR primer must be complementary to its template DNA to effectively amplify, the template with a certain point mutation can be distinguished from the normal template. The sensitivity of this detection method is higher than that of HRM, about 1% (CN104099422A). (3) Allele-specific Taqman polymerase chain reaction (CAST-PCR). CAST-PCR uses a specially designed MGB probe to prevent the primer from binding to wild-type DNA, and selectively and preferentially amplifies mutant DNA. The sensitivity of this detection method is about 0.1%-1% (Patent No. CN104099422A) . BarbanoR.etal applied CAST-PCR technology to detect V600E and V600K mutations of BRAF gene in clinical samples, and the sensitivity of the method was 1% (Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients).

To sum up, most of the existing gene mutation detection technologies are dye-based or probe-based fluorescence quantitative PCR technology, which has problems such as low sensitivity, low accuracy, complex positive interpretation methods, and high requirements for the type and quality of samples. For example, some methods require tissue samples, and some methods can process plasma/serum samples but require patients with stage III or IV tumors.

The invention uses digital PCR (digital PCR) technology to distribute a sample to tens of thousands of independent small droplets. signal for analysis. Digital PCR has the advantages of high sensitivity, accurate quantification without standard curve, and simple operation.

The present invention develops a high-sensitivity and high-specificity digital PCR method for EGFR S768I/EGFR L861Q/EGFR G719X (G719S, G719C, G719A) mutations, compared with the prior art HRM, Arms-qPCR, CAST-PCR, etc. The invention adopts Taqman probe combined with digital PCR method, which can solve the problems of low sensitivity, poor specificity, high requirements on the type and quality of samples, and complex positive interpretation methods. It has been verified that the sensitivity of the method of the present invention can reach a maximum of 0.06%, which can not only accurately detect tissue and body fluid samples, but also process difficult samples such as plasma/serum.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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<110> Ming Chi Biotechnology (Shanghai) Co., Ltd.

Mingshi Medical Technology (Ningbo) Co., Ltd.

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tccaggaagc ctacgtga 18

<210> 26

<211> 18

<212> DNA

<213> Artificial sequence

<400> 26

tgatgagctg cacggtgg 18

<210> 27

<211> 19

<212> DNA

<213> Artificial sequence

<400> 27

tccaggaagc ctacgtgat 19

<210> 28

<211> 18

<212> DNA

<213> Artificial sequence

<400> 28

ctgcacggtg gaggtgag 18

<210> 29

<211> 18

<212> DNA

<213> Artificial sequence

<400> 29

ccctccagga agcctacg 18

<210> 30

<211> 16

<212> DNA

<213> Artificial sequence

<400> 30

aggcagccga aggca 16

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<400> 31

aggaacgtac tggtgaaaac 20

<210> 32

<211> 21

<212> DNA

<213> Artificial sequence

<400> 32

tctgcatggt attctttctc t 21

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<400> 33

aacgtactgg tgaaaacacc 20

<210> 34

<211> 21

<212> DNA

<213> Artificial sequence

<400> 34

ctgcatggta ttctttctct t 21

<210> 35

<211> 18

<212> DNA

<213> Artificial sequence

<400> 35

accgcagcat gtcaagat 18

<210> 36

<211> 23

<212> DNA

<213> Artificial sequence

<400> 36

tccttctgca tggtattctt tct 23

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<400> 37

gcagcatgtc aagatcacag 20

<210> 38

<211> 21

<212> DNA

<213> Artificial sequence

<400> 38

cctccttctg catggtattc t 21

<210> 39

<211> 24

<212> DNA

<213> Artificial sequence

<400> 39

tgaggatctt gaaggaaact gaat 24

<210> 40

<211> 18

<212> DNA

<213> Artificial sequence

<400> 40

gtgccaggga ccttacct 18

<210> 41

<211> 22

<212> DNA

<213> Artificial sequence

<400> 41

gctctcttga ggatcttgaa gg 22

<210> 42

<211> 22

<212> DNA

<213> Artificial sequence

<400> 42

ggaccttacc ttatacaccg tg 22

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence

<400> 43

agctcccaac caagctctct 20

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence

<400> 44

cttaccttat acaccgtgcc 20

<210> 45

<211> 22

<212> DNA

<213> Artificial sequence

<400> 45

ctctcttgag gatcttgaag ga 22

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence

<400> 46

ttaccttata caccgtgccg 20

Claims (10)

1. A reagent for detecting a gene mutation, wherein the reagent is selected from the group consisting of:

(a) a first primer pair for detecting EGFR S768 mutation, wherein the first primer pair comprises primers shown as SEQ ID Nos. 1 and 2;

(b) a second primer pair for detecting EGFR L861 mutation, wherein the second primer pair comprises primers shown as SEQ ID Nos. 5 and 6;

(c) a third primer pair for detecting EGFR G719 mutation, wherein the third primer pair comprises primers shown as SEQ ID Nos. 9 and 10;

(d) combinations of (a), (b) and (c) above.

2. The reagent of claim 1, further comprising:

(a1) a first probe for use with a first primer pair, wherein said first probe is selected from the group consisting of: a probe shown as SEQ ID No. 3, a probe shown as SEQ ID No. 4, or a combination thereof.

3. The reagent of claim 1, further comprising:

(b1) a second probe for use with a second primer pair, wherein said second probe is selected from the group consisting of: the probe shown as SEQ ID No. 7, the probe shown as SEQ ID No. 8 or the combination thereof.

4. The reagent of claim 1, further comprising:

(c1) a third probe for use with a third primer pair, wherein said third probe is selected from the group consisting of: the probe shown as SEQ ID No. 11, the probe shown as SEQ ID No. 12, the probe shown as SEQ ID No. 13, the probe shown as SEQ ID No. 14 or the combination thereof.

5. The reagent of claim 2, wherein the first probe has the structure (5'-3') according to formula I:

Z1-Z2-Z3 I

wherein,

z1 is a fluorophore;

z2 is a specific complementary nucleic acid sequence with or without locked nucleotides;

z3 is a quencher group;

"-" is a bond, a linker, or a linker of 1-3 nucleotides.

6. The reagent of claim 3, wherein the second probe has the structure (5'-3') shown in formula II:

Z1'-Z2'-Z3'II

wherein,

z1' is a fluorophore;

z2' is a specific complementary nucleic acid sequence with or without locked nucleotides;

z3' is a quencher group;

"-" is a bond, a linker, or a linker of 1-3 nucleotides.

7. The reagent of claim 4, wherein the third probe has the structure (5'-3') shown in formula III:

Z1”-Z2”-Z3”III

wherein,

z1 "is a fluorophore;

z2' is a specific complementary nucleic acid sequence with or without locked nucleotides;

z3 "is a quencher group;

"-" is a bond, a linker, or a linker of 1-3 nucleotides.

8. A kit comprising the reagent for detecting a gene mutation according to claim 1.

9. Use of the reagent for detecting gene mutation of claim 1 or the kit of claim 8 for preparing a diagnostic product for pre-evaluating the effect of a subject on an EGFR-targeting drug.

10. A method for detecting whether a sample to be detected contains gene mutation is characterized by comprising the following steps:

(S1) providing a PCR reaction system, wherein the PCR reaction system contains a sample to be tested as a template and a primer pair for amplification, and the primer pair is selected from the group consisting of:

(a) a first primer pair for detecting EGFR S768 mutation, wherein the first primer pair comprises primers shown as SEQ ID Nos. 1 and 2;

(b) a second primer pair for detecting EGFR L861 mutation, wherein the second primer pair comprises primers shown as SEQ ID Nos. 5 and 6;

(c) a third primer pair for detecting EGFR G719 mutation, wherein the third primer pair comprises primers shown as SEQ ID Nos. 9 and 10;

(d) combinations of (a), (b), and (c) above;

a reagent for detecting a gene mutation according to claim 1;

(S2) performing a PCR reaction on the PCR reaction system of step (S1), thereby obtaining an amplification product;

(S3) analyzing the amplification product generated in the step (S2), thereby obtaining an analysis result of whether the test sample contains a gene mutation.

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