CN113930500B

CN113930500B - Digital PCR detection method and application of human PIK3CA gene mutation

Info

Publication number: CN113930500B
Application number: CN202010670563.7A
Authority: CN
Inventors: 江萤; 童慧娟; 黄昕
Original assignee: Mingshi Medical Technology Ningbo Co ltd; Mingchi Biotechnology Shanghai Co ltd
Current assignee: Mingshi Medical Technology Ningbo Co ltd; Mingchi Biotechnology Shanghai Co ltd
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2024-11-19
Anticipated expiration: 2040-07-13
Also published as: CN113930500A

Abstract

The invention provides a digital PCR detection method for human PIK3CA gene mutation and application thereof. Specifically, a primer pair, an amplification method, a nucleic acid probe and a detection system for sequences of PIK3CA E545, E542 and PIK3CA H1047 are provided, and a kit for detecting PIK3CA gene mutation is further provided. The invention can detect PIK3CA gene mutation with high sensitivity and strong specificity to different samples through optimized primer pairs, probes and corresponding reaction conditions.

Description

Digital PCR detection method for human PIK3CA gene mutation and application thereof

Technical Field

The invention relates to the field of gene detection, in particular to a digital PCR detection method for human PIK3CA gene mutation and application thereof.

Background

PIK3CA is a protooncogene located on chromosome 3, and has 20 exons in total. About 80% of PIK3CA mutations occur in the two hot spot regions of the helical region (Helical) and the Kinase region (Kinase), with the three most common mutations being H1047R on exon 20, E542K and E545K on exon 9. Mutation of the PIK3CA gene not only can cause enhancement of catalytic activity of PI3Ks, but also can promote canceration of cells.

Activation of PI3Ks as EGFR downstream signaling molecules can result in tumor cell resistance to EGFR-TKI drugs, e.g., mutation of the PIK3CA gene can result in resistance of cetuximab, panitumumab, to metastatic colorectal cancer treatment, resulting in resistance of gefitinib, erlotinib to non-small cell lung cancer treatment. PIK3CA mutations were ineffective for the breast cancer individualization targeting drug trastuzumab treatment, and wild-type treatment was effective. KRAS, BRAF, PIK3 any one or more mutations in CA were not effective in the treatment of colorectal cancer individuation targeting drug cetuximab, panitumumab, all wild-type treatments.

Therefore, detection of PIK3CA gene mutation can provide reference for doctors to select gefitinib, erlotinib, and icotinib and other targeted drug therapies in non-small cell lung cancer patients; treatment with targeted drugs such as cetuximab and panitumumab in colorectal cancer patients provides a reference; selection of targeted drug therapies such as trastuzumab in breast cancer patients provides a reference.

However, in the process of actually detecting the mutation of the PIK3CA gene of colorectal cancer patients, the problems of detecting the quality of samples and the like still remain. 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, abbreviated as "cfDNA"), also known as "liquid biopsy", in plasma avoids the need for biopsy of tumor tissue, and is a clinically very useful diagnostic application. The use of liquid biopsies provides the possibility of repeated blood sampling, allowing changes in cfDNA to be tracked during tumorigenesis or during cancer treatment, thereby monitoring changes in the condition.

However, there is a certain difficulty in using cell-free nucleic acids (such as cfDNA) as biomarkers in tumor patients, and especially the accurate and specific detection of gene mutations using cfDNA detection currently presents a great technical challenge. First, cfDNA content in blood varies from person to person and is very low in most cases, whereas the quality of free nucleic acid (Circulating tumor DNA, abbreviated as "ctDNA") of tumor origin is more ragged and of varying content.

Furthermore, cfDNA detection method specificity needs to be improved. Douillard et al report that the use of plasma to detect EGFR mutations is only 65% consistent with the results of tumor tissue detection.

In addition, although there are some methods for detecting PIK3CA gene mutation based on cfDNA, the sensitivity and specificity of these methods are not satisfactory yet.

Thus, there is an urgent need in the art to develop a method for detecting PIK3CA gene mutation based on cfDNA with high sensitivity and high specificity.

Disclosure of Invention

The invention aims to provide a method for detecting PIK3CA gene mutation with high sensitivity and high specificity based on cfDNA.

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

(a) A first primer pair for detecting a mutation in PIK3CA E545 and/or E542, wherein the first primer pair comprises the primers set forth in SEQ ID nos 1 and 2;

(b) A second primer pair for detecting a mutation in PIK3CA H1047, wherein the second primer pair comprises the primers shown in SEQ ID nos. 7 and 8;

(c) Combinations of (a) and (b) above.

In another preferred embodiment, the sequences of PIK3CA E545 and E542 are located in NG_01113.2:74725-74820.

In another preferred example, the sequence of the PIK3CA H1047 is located in NG_012113.2:90725-90819.

In another preferred embodiment, the nucleic acid sequence of the wild type sequence of PIK3CA E545/E542 is as shown in SEQ ID NO. 11.

In another preferred embodiment, the mutation is PIK3CA E545K, E542K, H1047R.

In another preferred embodiment, the nucleic acid sequence of the wild type sequence of PIK3CA H1047 is as shown in SEQ ID NO. 14.

In another preferred embodiment, the PIK3CA E545K mutation refers to a mutation of glutamic acid E at position 545 of the amino acid sequence of the PIK3CA protein to lysine K (i.e., E545K).

In another preferred embodiment, the PIK3CA E542K mutation refers to a mutation of glutamic acid E at position 542 of the amino acid sequence of the PIK3CA protein to lysine K (i.e., E542K).

In another preferred embodiment, the mutation of PIK3CA H1047R refers to a mutation of histidine H at position 1047 of the amino acid sequence of PIK3CA protein to arginine R (i.e. H1047R).

In another preferred embodiment, the PIK3CA E545K mutation refers to a mutation of guanine G at position 1633 of the nucleic acid sequence of the PIK3CA gene into adenine A (PIK 3CA c.1633G > A).

In another preferred embodiment, the mutation of PIK3CA E542K refers to the mutation of guanine G at position 1624 of the nucleic acid sequence of the PIK3CA gene into adenine A (PIK 3CA c.1624G > A).

In another preferred embodiment, the mutation of PIK3CA H1047R refers to the mutation of adenine A at position 3140 of the nucleic acid sequence of PIK3CA gene to guanine G (PIK 3CA c.3140A > G).

In another preferred embodiment, the PIK3CA E545K mutant nucleic acid sequence is as shown in SEQ ID NO. 12.

In another preferred embodiment, the PIK3CA E542K mutant nucleic acid sequence is as set forth in SEQ ID NO. 13.

In another preferred embodiment, the PIK3CA H1047R mutant nucleic acid sequence is as set forth in SEQ ID NO. 15.

In another preferred embodiment, the reagent further comprises:

(a1) A first probe for use with a 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, the probe shown in SEQ ID No. 5, the probe shown in SEQ ID No. 6, or a combination thereof.

In another preferred embodiment, the reagent further comprises:

(b1) A second probe for use with a second primer pair, wherein the second probe is selected from the group consisting of: the probe shown in SEQ ID No. 9, the probe shown in SEQ ID No. 10, or a combination thereof.

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

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

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

Z1-Z2-Z3 I

wherein,

Z1 is a fluorescent group;

Z2 is a specific complementary nucleic acid sequence;

z3 is a quenching group;

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

In another preferred embodiment, the Z2-specific nucleic acid sequence targets wild-type PIK3CA E545, E542 sites.

In another preferred embodiment, the Z2-specific nucleic acid sequence targets the mutant PIK3CA E545K site.

In another preferred embodiment, the Z2-specific nucleic acid sequence targets the mutant PIK3CA E542K site.

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

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

TCTCCTGCTCAGTGA(SEQ ID No:3)；

TCTCCTGCTTAGTGA(SEQ ID No:4)；

CGAGATCCTCTCTCTGAA(SEQ ID No:5)；

CGAGATCCTCTCTCTAAAA(SEQ ID No:5)。

in another preferred embodiment, the fluorophores are each independently located at the 5 'end, the 3' end, and the middle of the nucleic acid probe.

In another preferred embodiment, the fluorophore and the quencher are each independently located at the 5 'end, the 3' end, and/or the middle portion.

In another preferred embodiment, the fluorophore comprises a fluorophore that is cross-linked to a DNA probe.

In another preferred embodiment, the fluorophore 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, BHQ 1, BHQ 2, BHQ3, MGB, NFQ, BBQ-650, TQ1-TQ6, QSY 7carboxyl ic acid, TQ7, eclipse, or a combination thereof.

In another preferred embodiment, the nucleic acid probe is WTP-PIK3CA E545 (SEQ ID NO: 3).

In another preferred embodiment, the nucleic acid probe is WTP-PIK3CA E545K (SEQ ID No: 4).

In another preferred embodiment, the nucleic acid probe is MTP-PIK3CA E542 (SEQ ID No: 5).

In another preferred embodiment, the nucleic acid probe is WTP-PIK3CA E542K (SEQ ID NO: 6).

In another preferred embodiment, the structure (5 '-3') of the second probe is represented by formula II:

Z1'-Z2'-Z3' I I

wherein,

Z1' is a fluorescent group;

Z2' is a specific complementary nucleic acid sequence;

Z3' is a quenching group;

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

In another preferred embodiment, the Z2' specific nucleic acid sequence targets the wild-type PIK3CA H1047 site.

In another preferred embodiment, the Z2' specific nucleic acid sequence targets the mutant PIK3CA H1047R site.

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

CACCATGATGTGCATCA(SEQ ID No:9)；

CACCATGACGTGCATC(SEQ ID No:10)。

In another preferred embodiment, the nucleic acid probe is WTP-PIK3CA H1047 (SEQ ID NO: 9).

In another preferred embodiment, the nucleic acid probe is MTP-PIK3CA H1047R (SEQ ID No: 10).

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

In another preferred embodiment, the kit further comprises a first probe for use with the first primer pair; and/or a second probe for use with a second primer pair.

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

In another preferred embodiment, the kit comprises a first primer pair as shown in SEQ ID Nos. 1 and 2, and a first probe as shown in SEQ ID Nos. 3, 4, 5 and 6.

In another preferred embodiment, the kit comprises a second primer pair as shown in SEQ ID Nos. 7 and 8, and a second probe as shown in SEQ ID Nos. 9 and 10.

In a third aspect of the invention there is provided the use of a reagent for detecting a mutation in a gene as described in the first aspect or a kit as described in the second aspect for the preparation of a diagnostic product for assessing whether a subject is suitable for treatment with an EGFR-targeting agent or for pre-assessing the effect of a subject using an EGFR-targeting agent.

In another preferred embodiment, the EGFR-targeting agent is selected from the group consisting of: non-tinib, erlotinib, icotinib, cetuximab, panitumumab, or a combination thereof.

In another preferred embodiment, the product is tested against cfDNA samples.

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

In another preferred embodiment, the subject is a tumor patient, and/or a leukemia 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: intestinal cancer, liver cancer, lung cancer, gastric cancer, breast cancer, or a combination thereof.

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

(S1) providing a PCR reaction system, wherein the PCR reaction system comprises a sample to be detected serving as a template and a primer pair for amplification, and the primer pair is selected from the following groups:

(c) A combination of (a) and (b) above;

The reagent for detecting a gene mutation according to the first aspect;

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

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

In another preferred embodiment, the analysis results are qualitative results.

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 in the microdroplet is 1 to 1X 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 is 56.+ -. 2 ℃, preferably 56.+ -. 1 ℃, more preferably 56.+ -. 0.5 ℃.

In another preferred embodiment, the PCR reaction system further comprises:

(a1) A first probe for use with a first primer pair, wherein the first probe is selected from the group consisting of: a probe shown in SEQ ID No. 3, a probe shown in SEQ ID No. 4, a probe shown in SEQ ID No. 5, a probe shown in SEQ ID No. 6, or a combination thereof; and/or

In another preferred embodiment, the probes (SEQ ID No:3, SEQ ID No:5 and SEQ ID No: 9) for detecting the wild-type gene employ the same first fluorescent label (e.g., HEX, FAM).

In another preferred embodiment, the probes (SEQ ID NOS: 4, 6 and 10) for detecting mutant genes employ the same second fluorescent label (e.g., HEX, FAM), and the first fluorescent label and the second fluorescent label are different.

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 method has a detection accuracy of 0.06% -1%, preferably 0.0625% -0.08%.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the result of PCR electrophoresis of the pair of primers PIK3CA E545 and E542 5 (M: DNA MARKER)

FIG. 2 shows the result of PCR electrophoresis of the PIK3CA H1047 5 pair primer (M: DNA MARKER)

FIG. 3 shows the annealing temperature optimization of Taqman-ddPCR method for detecting the E545K mutation of the PIK3CA gene, and the pore B\D\F\H represents the annealing temperature gradient of 60 ℃, 58 ℃, 56 ℃ and 54 ℃. Wherein A is a 1-D amplitude plot of FAM channel detection positive control (100% E545K mutation); b is HEX channel detection negative control 1-D amplitude plot

Fig. 4: shows the annealing temperature optimization of Taqman-ddPCR method for detecting the E542K mutation of PIK3CA gene, and the holes A\C\E\G represent the annealing temperature gradient of 60 ℃, 58 ℃, 56 ℃ and 54 ℃. Wherein A is a 1-D amplitude plot of FAM channel detection positive control; b is HEX channel detection positive control 1-D amplitude plot.

FIG. 5 shows the annealing temperature optimization of Taqman-ddPCR method for detecting the mutation of the H1047R gene of PIK3CA, and the columns of holes A\C\E\G represent the annealing temperature gradient of 60 ℃, 58 ℃, 56 ℃ and 54 ℃. Wherein A is a 1-D amplitude plot of FAM channel detection positive control; b is HEX channel detection positive control 1-D amplitude plot.

FIG. 6 shows the verification of the detection concentration of the PIK3CA E545K gene mutation. As can be seen from the graph, the linearity of the detection method in all detection concentrations (0.06% -1%) is very good, i.e. the sensitivity of the method reaches 0.06%

FIG. 7 shows the verification of the detection concentration of the PIK3CA E542K gene mutation. The graph shows that the linearity of the detection method in all detection concentrations (0.06% -1%) is very good, namely the sensitivity of the method reaches 0.06%.

FIG. 8 shows the verification of the detection concentration of the mutation in the PIK3CA H1047R gene. The graph shows that the linearity of the detection method in all detection concentrations (0.06% -1%) is very good, namely the sensitivity of the method reaches 0.06%.

FIG. 9 shows that case "MS1106" was detected as PIK3CA negative by digital PCR. From top to bottom, the two-dimensional plot of the detection (2D amplitude plot of FAM-HEX channel) was of E545K, E542K, H1047R, respectively. Wherein green is the wild type signal.

FIG. 10 shows a two-dimensional map of the case "MS1124" detected by digital PCR detection PIK3CA E545K (2D map of FAM-HEX channel). Wherein blue and orange are mutation signals, indicating that case "MS1124" was PIK3CA E545K positive by digital PCR detection.

FIG. 11 shows a two-dimensional map of the case "MS1133" detected by digital PCR detection PIK3CA E542K (2D map of FAM-HEX channel). Wherein blue and orange are mutation signals indicating PIK3CA E542K positive by digital PCR detection.

FIG. 12 shows a two-dimensional map of the case "MS1127" detected by digital PCR detection PIK3CA H1047R (2D map of FAM-HEX channel). Wherein blue and orange are mutation signals, indicating positive PIK3CA H1047R by digital PCR detection.

Detailed Description

Through extensive and intensive research, the inventor can effectively improve the gene mutation detection effect by a large number of screening, particularly optimizing primer sequences and probes and combining with a digital PCR platform, breaks through the problems of low accuracy, low sensitivity and the like of pathological tissues serving as starting materials in the existing gene mutation detection technology, and provides a method for detecting PIK3CA gene mutation with high specificity, high sensitivity and strong anti-interference capability. On this basis, the present inventors have completed the present invention.

Specifically, the invention provides a detection method for PIK3CA E545K, PIK3CA E542K and PIK3CA H1047R mutant genes, namely a digital PCR detection system is established by providing 2 pairs of primer pairs which are specially optimized for sequences in which PIK3CA E545/E542 and PIK3CA H1047 are located and probes which are specially optimized for PIK3CA E545K, PIK CA E542K, PIK CA H1047R mutant genes, so that the mutation situation of the PIK3CA genes can be qualitatively and quantitatively detected. The method and the reagent provided by the invention have unexpectedly high sensitivity and high specificity when being used for detecting PIK3CA gene mutation, and can detect samples with different difficulties.

Terminology

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

The term "about" may refer to a value or composition that is within an acceptable error 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 measured. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

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

Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a well known method to those skilled in the art.

Digital PCR (digital PCR) technology

The technique of number PCR (digital PCR), which is based on single molecule PCR method to perform quantitative nucleic acid counting, is an absolute quantitative method. The method mainly adopts a microfluidic or microdroplet method in the current analytical chemistry hot research field to disperse a large amount of diluted nucleic acid solution into micro-reactors or microdroplets of a chip, wherein the number of nucleic acid templates in each reactor is less than or equal to 1. After the PCR cycle, the fluorescent signal of each droplet is analyzed after the amplification is finished, the reactor with the nucleic acid molecular template gives out the fluorescent signal, and the reactor without the template has no fluorescent signal. From the relative proportions and the volume of the reactor, the nucleic acid concentration of the original solution can be deduced.

Digital PCR is capable of accurately quantifying and detecting a target nucleic acid molecule with high sensitivity, compared to conventional qPCR. The method of analyzing the result of conventional qPCR is an analog method in which the digital PCR method, the result of which is analyzed by a digital method (because the resulting signal has a value of "0" or "1"), has the advantage that a large volume of sample can be analyzed, different samples can be detected simultaneously, and different tests can be performed simultaneously. Digital PCR technology is a technology that can absolutely quantify DNA samples using single molecule counting without standard curves, and can more accurately absolute quantify individual droplets per well by PCR (see Gudrun Pohl and le-mig Shih, principles and applications of digital PCR (PRINCIPLE AND applications of DIGITAL PCR), expert rev.mol.diagn.4 (1), 41-47 (2004)). The digital PCR has the advantages of high sensitivity, accurate quantification without a standard curve, simple operation and the like.

In digital PCR, each droplet containing a sample gene template, amplification primers and fluorescent probes prepared so as to be available for dilution to an average copy number of 0.5-1 is dispensed into a single well, and microemulsion PCR is performed. Then, the well count showing the fluorescent signal was a value of "1", because the sample having the gene copy number of 1 was allocated to the well and showed the fluorescent signal after amplification, whereas the well count showing no signal was a value of "0", because the sample having the gene copy number of 0 was allocated to the well, and showed no fluorescent signal due to no amplification. In this way, absolute quantification can be achieved.

Primer(s)

A primer refers to a macromolecule with a specific nucleotide sequence that stimulates synthesis at the initiation of nucleotide polymerization, and is covalently linked to a reactant. Primers are typically two oligonucleotide sequences that are synthesized, one complementary to one strand of the DNA template at one end of the target region and the other complementary to the other strand of the DNA template 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 fragment in the detection system is amplified in advance, and thus a primer corresponding to the sequence in which the mutation is located is designed.

In a preferred embodiment, aiming at the sequence of the PIK3CA E545/E542, a plurality of pairs of primers are designed at the upstream and downstream of the E545/E542 locus of the PIK3CA gene, and the optimal primer pairs are finally determined to be SEQ ID No. 1 and 2 through experimental tests.

In another preferred example, aiming at the sequence of the PIK3CA H1047, a plurality of pairs of primers are respectively designed at the upstream and downstream of the H1047 locus of the PIK3CA gene, and the optimal primer pairs are finally determined to be SEQ ID No. 3 and 4 through experimental tests.

Probe with a probe tip

As used herein, "probe," "nucleic acid probe," "genetic probe" may be used interchangeably to refer to a nucleic acid sequence (DNA or RNA) with a detectable label and known in sequence that is complementary to a gene of interest. The gene probe is combined with the target gene through molecular hybridization to generate hybridization signals, and the target gene can be displayed from a vast genome. According to the hybridization principle, the nucleic acid sequence as a probe must have at least the following two conditions: ① If the double strand is used, the double strand must be denatured; ② Should be provided with a label that is easily detectable. The nucleic acid probe may include the entire gene or may be only a part of the gene; either DNA itself or RNA transcribed from it. In the present invention, the probe also refers to a modified primer, wherein both ends or the middle of the modified primer are provided with chemical modification groups, and the chemical modification has special functions including but not limited to: signaling, enhancing the attachment to the reactants, etc.

The structure of the first probe is shown as a formula I:

Z1-Z2-Z3 I

wherein,

Z1 is a fluorescent group;

Z2 is a specific complementary nucleic acid sequence;

z3 is a quenching group;

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

TCTCCTGCTCAGTGA(SEQ ID No:3)；

TCTCCTGCTTAGTGA(SEQ ID No:4)；

CGAGATCCTCTCTCTGAA(SEQ ID No:5)；

CGAGATCCTCTCTCTAAAA(SEQ ID No:5)。

In another preferred embodiment, the quenching group is selected from the group consisting of: DABCYL, TAMRA, BHQ 1, BHQ 2, BHQ3, MGB, NFQ, BBQ-650, TQ1-TQ6, QSY 7 carboxilic acid, TQ7, eclipse, or a combination thereof.

The structure (5 '-3') of the second probe is shown as a formula II:

Z1'-Z2'-Z3' II

wherein,

Z1' is a fluorescent group;

Z2' is a specific complementary nucleic acid sequence;

Z3' is a quenching group;

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

CACCATGATGTGCATCA(SEQ ID No:9)；

CACCATGACGTGCATC(SEQ ID No:10)。

Modification of primers and probes

In the present invention, the nucleic acid sequence of the primer includes an unmodified or modified primer sequence.

Preferably, the present inventors have significantly improved the specificity of the probe by modifying the probe with a locked nucleotide, thereby improving the sensitivity and specificity of the detection result.

In a preferred embodiment of the invention, the modification is carried out in a manner selected from the group consisting of: phosphorylation (Phosphorylation), biotin (Biotin), digoxigenin (Digoxigenin), internal amino modification, 5 'amino modification, 3' amino modification, sulfhydryl group (thio), spacer (Spacer), thio (Phosphorthioate), deoxyuracil (DeoxyUridine, dU), deoxyhypoxanthine (deoxyInosine, dI), or combinations thereof.

Phosphorylation modification: 5' phosphorylation can be used for ligation reactions catalyzed by adaptors, cloning and genetic constructs, and ligases. In related experiments where 3 'phosphorylation was resistant to 3' exonuclease digestion, it was also used to prevent DNA polymerase catalyzed DNA chain extension reactions.

Modification of biotin: the primer biotin label can be used for detecting proteins, intracellular chemical staining, cell separation, nucleic acid separation, hybridization detection specific DNA/RNA sequences, ion channel conformational changes and the like by non-radioactive immunoassay.

And (3) high and new modification: the cadaverine is linked to the C5 position of uracil via an 11 atom spacer, and hybridized cadaverine probes can be detected by anti-cadaverine antibodies. The labeled probe can be used in various hybridization reactions such as DNA-DNA hybridization (Southern blotting), DNA-RNA hybridization (Northern blotting), dot blotting, cloning hybridization, in situ hybridization, and enzyme-linked immunosorbent assay (ELISA).

Modification of internal amino: the internal modification is mainly performed by adding C6-dT aminolinker to thymine residues. The modified amino groups are 10 atoms away from the backbone and can be used for further labeling and enzymatic ligation (e.g., alkaline phosphatase), currently providing internal amino modification-mediated dT-Dabcyl, dT-Biotin and dT-Digoxingenin modifications.

5' Amino modification: can be used for preparing functionalized oligonucleotides, and can be widely applied to DNA chips (DNA microarrays) and multiple-marker diagnostic systems. Currently, both 5'C6 amino modifications are provided, which can be used to attach compounds that do not affect the function of the oligonucleotide even when in close proximity thereto, and 5' C12 amino modifications, which can be used to attach affinity purification groups and to label with some fluorescence, especially when the fluorescence may be quenched by the label being too close to the DNA strand.

3' Amino modification: currently 3' c6 amino modifications are provided. 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' end can be modified with an amino group for additional ligation. In addition, 3' modification can inhibit 3' exonuclease enzymolysis, so that the modified 3' exonuclease can be used for antisense experiments.

Thiol modification: the 5' -mercapto group is similar in many respects to the amino modification. Sulfhydryl groups can be used to attach various modifications such as fluorescent labels and biotin. For example, thiol-linked fluorescent probes can be made in the presence of iodoacetic acid and maleimide derivatives. Thiol-modification of 5' the monomers are modified predominantly with 5' Thiol (5 ' -thio-Modifier C6-CE Phosphoramidite or thio-Modifier C6S-S CE Phosphoramidite). Silver nitrate oxidation is necessary to remove the protecting group (trityl) after modification with the 5' -thio-Modifier C6-CE monomer, while DTT is necessary to reduce the disulfide bond to a Thiol group after modification with the thio-Modifier C6S-S CE monomer.

Modification of a spacer arm: the Spacer can provide the necessary spacing for oligonucleotide labeling to reduce interactions between the labeling groups and the oligonucleotides, and is mainly applied to DNA hairpin structure and double-stranded structure research. C3 The spacer is used primarily to mimic the three carbon spacing between the 3 'and 5' hydroxyl groups of ribose, or "substitute" for 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 acting. Spacer 18 is often used to introduce a strong hydrophilic group.

Thio modification: thio-modified oligonucleotides are mainly used in antisense experiments to prevent degradation by nucleases. However, as the number of thioated bases increases, the Tm of the oligonucleotide decreases, and in order to reduce this effect, 2 to 5 bases at both ends of the primer may be thio-modified, and usually 3 bases at each of 5 'and 3' may be thio-modified.

Deoxyuracil modification: deoxyuracils can be inserted into the oligonucleotides to increase the melting point temperature of the duplex and thus increase the stability of the duplex. Replacement of each deoxythymine by a deoxyuracil increases the temperature of the melting point of the duplex by 1.7 ℃.

Deoxyhypoxanthine modification: deoxyinosine is a naturally occurring base that, although not truly universal, is relatively more stable when bound to other bases than other base mismatches. The binding capacity of deoxyinosine with other bases is dI/dC > dI/dA > dI/dG > dI/dT. under the catalysis of DNA polymerase, and deoxyinosine is combined with dC preferentially.

cfDNA

Free nucleic acid (Circulating free DNA, abbreviated as "cfDNA") in plasma, also known as "liquid biopsy", avoids the need for biopsies of tumor tissue, and is a clinically very useful diagnostic application. The use of liquid biopsies provides the possibility of repeated blood sampling, allowing changes in cfDNA to be tracked during tumorigenesis or during cancer treatment, and thus monitoring for changes in conditions (Cell-free nucleic acids as biomarkers IN CANCER PATIENTS). However, the use of cfDNA detection to accurately and specifically detect gene mutations currently presents a significant technical challenge. First, cfDNA content in blood varies from person to person and is very low in most cases, whereas free nucleic acid (Circulating tumor DNA, abbreviated as "ctDNA") of tumor origin is of varying quality and content. Furthermore, cfDNA detection method specificity needs to be improved. Douillard et al reports that the compliance of EGFR mutations detected using plasma with tumor tissue detection results was only 65%.

The main advantages of the invention include:

1. The sensitivity is high: because the method adopts a digital PCR platform, the reaction system can be divided into about 20000 micro reactions, and single copy mutation can be detected theoretically, so that the method has the sensitivity advantage that other technologies cannot match. The detection method provided by the invention can reach the minimum detection limit of 0.06% through verification.

2. The specificity is strong: the designed specific primers are respectively aimed at sequences of PIK3CA genes E545 and E542 and PIK3CA gene H1047, and can specifically amplify wild type and mutant templates at target positions; the designed specific probe covers mutation sites, 2 wild type (total 2) and mutant type (total 3) of PIK3CA are designed respectively, HEX fluorescent groups are modified at the 5' end of the wild type probe, FAM (FAM) should be modified at the 5' end of the mutant type probe, meanwhile, the PIK3CA probe is modified by Locked Nucleic Acid (LNA) at the position of a mutant base, the binding force of the nucleotide at the position is greatly enhanced, the PIK3CA probe is provided with BHQ 1at the 3' end, templates with one base difference can be effectively distinguished, and the design of the primers and the probes greatly improves the detection specificity.

3. The requirements on the type and the quality of the sample are relaxed, and the anti-interference capability is strong. Because of the high sensitivity, the invention is applicable to the type of samples, which can be used for peripheral blood samples (the samples are easy to obtain, but have low DNA content and are broken) besides fresh tissue samples and paraffin sections which are commonly used in the common method; moreover, due to the uniqueness of the digital PCR platform, the reaction system can be divided into about 20000 small systems, and meanwhile, the interference substances can be divided into about 20000 parts, so that the influence of the interference substances on the reaction can be greatly reduced, and samples with more complex backgrounds can be detected. This is not possible with other platforms.

4. The positive interpretation method is simple: because the absolute quantification method is adopted in the invention, a comparison standard curve is not required to be set, and whether the target mutation template is contained can be judged according to a two-dimensional fluorescent graph as a result (table 1).

TABLE 1 detection results Table

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: coldSpringHarborLaboratoryPress, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

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

Primer, probe and amplified fragment sequences

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

table 2: primer, probe and nucleic acid sequence of DNA fragment

Example 1 screening of primer pairs

Based on the exon 9 sequences of PIK3CA (E545, E542 sites), 5 primer pairs (primers were synthesized from Shanghai Biochemical Co., ltd.) were designed and the specific sequences are shown in Table 3 below.

TABLE 3 screening of 5 primer pairs for amplification of PIK3CA E545, E542

Based on the 20 th exon sequence of PIK3CA (H1047 site), 5 pairs of primers (primer was synthesized from Shanghai Biochemical Co., ltd.) were designed, and the specific sequences are shown in Table 4 below.

TABLE 4 screening of 5 primer pairs for amplification of PIK3CA H1047

The PCR system was selected from the group consisting of 0.2ul of Taq HS polymerase, 2ul of 10 XHS 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 make-up system to 20ul.

Screening PCR procedure: 95℃for 10min,30 cycles (94℃for 30s,55℃for 30s,72℃for 20 s).

Through PCR screening, the E545 and E542 primer pair 2 (E545/E542-F2 and E545/E542-R2) and the H1047 primer pair 2 (H1047-F2/H1047-R2) have higher amplification efficiency (bright stripes) on target fragments, have shorter amplified products, do not generate primer dimers, have the best overall effect, and can be used as primer pairs for PIK3CA gene mutation detection. See fig. 1 and 2.

Example 2 Taqman optimization of probes and digital PCR reaction procedure

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

2.1 Taqman probe

In this example, the PIK3CA gene mutation detected was a nucleotide mutation corresponding to E545K, E542K, H1047R (table 5);

Table 5: PIK3CA gene mutation information table

Detection site	Mutation type	COSMIC numbering	Gene change
				E545K	Point mutation	COSM763	c.1633G>A
E542K	Point mutation	COSM760	c.1624G>A
				H1047R	Point mutation	COSM775	c.3140A>G

Based on the genome sequence and mutation site of human PIK3CA, designing corresponding Taqman probe and optimizing, wherein the optimized Taqman probe contains specific locked nucleotide (+N) which is WTP-E545/MTP-E545K, WTP-E542/MTP-E542K, WTP-H1047/MTP-H1047R, and specific information of the probe is shown in Table 2.

2.2 Optimization of digital PCR reaction program

Further optimizations were made to the digital PCR reaction procedure based on the optimal primer pairs determined in example 1 (SEQ ID Nos: 1 and 2; SEQ ID Nos: 7 and 8) and the optimized probes determined in example 2.1. The digital PCR reaction program optimization experiment needs to comprehensively analyze the digital PCR amplification conditions of the wild type template and the mutant template along with the change of the annealing temperature. The optimal annealing temperature should satisfy two conditions: negative control (wild-type template) background was clean (no droplet on FAM channel), positive and negative signals were easily distinguished in mutant templates (fluorescence intensity of FAM channel and HEX channel were high).

The experimental method is as follows:

The following PCR system was arranged in the reagent preparation area: 2 XddPCR Supermix (No dUTP) 11ul, upstream primer (E545/E542-F or H1047-F) 1.1ul, downstream primer (E545/E542-R or H1047-R) 1.1ul, probe 1 (WTP-E545, WTP-E542 or WTP-H1047) 0.55ul, probe 2 (MTP-E545K or MTP-H1047R) 0.55ul, template 5.5ul, and water was added to fill up to 22ul.

The templates were added to the sample preparation zone in the following order: negative control, positive control. The negative control is normal human leukocyte genome (tgDNA), and the positive control is genomic DNA solution containing PIK3CA corresponding mutation sequence.

In the droplet generation zone, droplet generation is performed as required by the instrument.

In the sample analysis zone, a PCR reaction is performed and analyzed.

PCR procedure for ddPCR optimization experiment: 95℃for 10min,40 cycles {94℃for 30s, annealing temperature gradient (60 ℃, 58 ℃, 56 ℃, 54 ℃) for 15s,72 ℃ for 15s },98℃for 10min, and heating and cooling rate of 2 ℃/s.

After the PCR is completed, setting is carried out according to the instrument and experimental requirements, FAM/HEX detection channels are selected, and reading of the plate is started.

The results are shown in FIGS. 3 to 5. Wherein the FAM channel indicates the MTP probe to detect the droplet of mutant template and its fluorescence intensity (amplide), and the HEX channel indicates the WTP probe to detect the droplet of wild-type template and its fluorescence intensity (amplide). Double positive microdroplets of FAM-HEX represent the presence of both wild-type and mutant templates. The number of droplets through the FAM channel can be used to infer the presence of mutant templates in the digital PCR system and to accurately calculate the copy number (copies/ul).

The results of FIGS. 3-5 show that the annealing at 56 ℃ for 15s, the digital PCR negative control has no pollution, the negative signal and the positive signal are most obviously distinguished, the overall effect is better, and the annealing temperature is the optimal annealing temperature.

EXAMPLE 3 digital PCR method sensitivity verification for detecting PIK3CA Gene mutation

Wild-type template copy number (copies/ul), mutant template genome copy number (copies/ul) and mutation rate (%) were calculated according to example 1, both diluted to 4000copies/ul with TE. The genome template containing the mutation is doped with tgDNA so that the ratio of the mutation template is 0.06%, 0.13%, 0.25%, 0.5% and 1% respectively, and the gradient dilution template is manufactured.

Verifying a digital PCR system: 2 XddPCR Supermix (No dUTP) 11ul, primer 1 (E545/E542-F, H1047-F) 1.1ul, primer 2 (E545/E542-R, H1047-R) 1.1ul, probe 1 (WTP-E545, E542/WTP-H1047) 0.55ul, probe 2 (MTP-E545K, MTP-E545K, MTP-H1047R) 0.55ul, template 5.5ul, and water was added to make up to 22ul.

The templates were added to the sample preparation zone in the following order: blank control, negative control, gradient dilution template. The blank control was water, the negative control was tgDNA, and the gradient dilution template was a wild-type template incorporating 0.06% -1% mutant template.

The PCR reaction system was carried out to generate a droplet in the same manner as in example 1. PCR was performed according to the optimized PCR procedure: 95℃for 10min,40 cycles (94℃for 30s,56℃for 15s,72℃for 15 s), 98℃for 10min, and a ramp rate of 2℃per second. And starting reading the plate according to the instrument requirement.

The results of the digital PCR are shown in FIGS. 6 to 8. The digital PCR method uses water or tgDNA as a template, and has clean background and no pollution. In addition, the digital PCR method of the invention is used for doping 0.06% -1% mutant template into wild template, detecting that the copy number of wild template is normal, detecting that the proportion of mutant template (the mutant template divided by the total template multiplied by 100%) is in accordance with the theoretical proportion and is in linear correlation, and the R ² value is more than 0.98. The digital PCR method is within the sensitivity interval of 0.06% -1%, the detection value is accurate, and the minimum detection sensitivity of the digital PCR method is 0.06%.

EXAMPLE 4 digital PCR method for detecting PIK3CA mutation in patients to guide tumor targeting drug administration

Cases "MS1106", "MS1124", "MS1133", "MS1127" are leukemia patients. Plasma was separated by two centrifugation steps by collecting venous blood and plasma free nucleic acid was extracted by the free nucleic acid extraction kit. The extracted nucleic acid was quantified by Qubit. After quantification, the sample was stored in a-20℃refrigerator.

The digital PCR detection system of the present invention is described in example 2.

The templates were added to the sample preparation zone in the following order: blank control, negative control, case free nucleic acid, positive control (1%).

The PCR reaction system was carried out to generate a droplet in the same manner as in example 1. PCR was performed according to the optimized PCR procedure: 95℃for 10min,40 cycles (94℃for 30s,56℃for 15s,72℃for 15 s), 98℃for 10min, and a ramp rate of 2℃per second. Opening the instrument, setting according to the requirement, and starting reading the board. The PIK3CA detection is clean with blank control and negative control background, and the copy number and mutation proportion of the positive control are normal.

The results of the case nucleic acid sample testing are shown in Table 6 below.

MS1106 judged negative (see FIG. 9), without E545K, E542 and H1047R mutations, and other gene mutations can be detected continuously for further medication guidance.

MS1124 judged E545K positive (see FIG. 10; mutation rate 0.743%), treatment with targeted drugs such as gefitinib, erlotinib, etc. was not suggested;

Case MS1133 interpreted as E542K positive (see FIG. 11; mutation rate 0.827%), no targeted drug therapy with gefitinib, erlotinib, etc. was suggested;

Case MS1127 interpreted as positive for H1047R (see FIG. 12; mutation rate 0.398%), and no targeted drug therapy with gefitinib, erlotinib, icotinib, etc. was suggested. Specific examination data for four cases are shown in the following table.

Specific test data for the above cases are shown in table 6 below:

Table 6: case detection Gene mutation results

Discussion:

Currently, the methods for detecting gene mutation mainly comprise:

(1) High resolution melting curve (HRM). HRM is a genetic analysis technique that forms melting curves of different forms based on the difference in melting temperature of single nucleotides. The detection sensitivity is about 1-10%. However, due to the high false positives of the dye method, sequencing verification is required in the later stage, resulting in a longer detection period.

(2) The probe amplification blocking mutation method (ARMS-qPCR). The template with a certain point mutation is distinguished from a normal template by utilizing the principle that the 3' -terminal base of the PCR primer must be complementary with the template DNA so as to effectively amplify, and the sensitivity of the detection method is higher than that of the HRM and is about 1 percent (CN 104099422A). (3) Allele specific Taqman polymerase chain reaction (CAST-PCR). CAST-PCR prevents the primer from combining with wild type DNA by a segment of specifically designed MGB probe, selectively and preferentially amplifies mutant DNA, and the sensitivity of the detection method is about 0.1% -1% (patent No. CN 104099422A). BarbanoR.et al uses CAST-PCR technology to detect V600E and V600K mutation of clinical sample BRAF gene, and the sensitivity of the method is as follows 1％(Competitive allele-specific TaqMan PCR(Cast-PCR)is a sensitive,specific and fast method for BRAF V600 mutation detection inMelanoma patients).

In summary, most of the existing genetic mutation detection technologies are dye-method or probe-method fluorescent quantitative PCR technologies, which have the problems of low sensitivity, low accuracy, complex positive interpretation method and the like, and have high requirements on the type and quality of samples, for example, some methods require providing tissue samples, and some methods can process plasma/serum samples but require patients in the third or fourth stage of tumor.

The invention uses the digital PCR (digital PCR) technology to distribute a sample to tens of thousands of independent small droplets, each droplet respectively carries out PCR amplification on target molecules, and fluorescent signals of each droplet are analyzed after the amplification is finished. The digital PCR has the advantages of high sensitivity, accurate quantification without a standard curve, simple operation and the like.

Aiming at PIK3CA E545K, E542K, H1047R mutation, the invention develops a high-sensitivity and high-specificity digital PCR method, and compared with the prior art of HRM, arms-qPCR, CAST-PCR and the like, the invention adopts a Taqman probe and combines the digital PCR method, thereby solving the problems of low sensitivity, poor specificity, high requirements on the type and quality of samples, complex positive interpretation method and the like. The sensitivity of the method can reach 0.06% at most, so that not only can tissue and body fluid samples be accurately detected, but also samples with high difficulty such as blood plasma/serum can be processed.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Ming's biological technology (Shanghai) Co., ltd

Nameplate medical science and technology (Ningbo) Limited

Digital PCR detection method of <120> human PIK3CA gene mutation and application thereof

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Claims

1. A reagent for detecting gene mutation, characterized in that the reagent is selected from the following group:

(a) a first primer pair for detecting PIK3CA E545 and/or E542 mutations, wherein the first primer pair comprises primers shown in SEQ ID Nos: 1 and 2; and

(b) a second primer pair for detecting PIK3CA H1047 mutation, wherein the second primer pair comprises primers shown in SEQ ID No: 7 and 8;

Wherein, the reagent also includes:

(a1) a first probe used in conjunction with the first primer pair, wherein the first probe is a combination of the following probes: a probe shown in SEQ ID No: 3, a probe shown in SEQ ID No: 4, a probe shown in SEQ ID No: 5, and a probe shown in SEQ ID No: 6; and

(b1) A second probe used in combination with the second primer pair, wherein the second probe is a combination of the following probes: a probe shown in SEQ ID No: 9 and a probe shown in SEQ ID No: 10.

2. The reagent according to claim 1, characterized in that the structure 5'-3' of the first probe is as shown in Formula I:

Z1-Z2-Z3 I

in,

Z1 is a fluorescent group;

Z2 is a specific complementary nucleic acid sequence;

Z3 is a quenching group;

“-” is a chemical bond, a linker, or a linker consisting of 1-3 nucleotides.

3. The reagent as claimed in claim 2, characterized in that the target of the specific complementary nucleic acid sequence is selected from the following group: wild-type PIK3CA E545, E542 site, mutant PIK3CA E545K site, mutant PIK3CA E542K site.

4. The reagent according to claim 2, wherein the sequence of the specific complementary nucleic acid is selected from the group consisting of:

TCTCCTGCTCAGTGA, SEQ ID No: 3;

TCTCCTGCTTAGTGA, SEQ ID No: 4;

CGAGATCCTCTCTCTgAA, SEQ ID No: 5;

CGAGATCCTCTCTCTAaaa, SEQ ID No: 6.

5. The reagent of claim 2, wherein 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.

6. The reagent of claim 2, wherein the quenching group is selected from the group consisting of DABCYL, TAMRA, BHQ1, BHQ 2, BHQ3, MGB, NFQ, BBQ-650, TQ1-TQ6, QSY 7carboxylic acid, TQ7, eclipse, or a combination thereof.

7. The reagent according to claim 1, characterized in that the structure 5'-3' of the second probe is as shown in Formula II:

Z1'-Z2'-Z3'II

in,

Z1' is a fluorescent group;

Z2' is a specific complementary nucleic acid sequence;

Z3' is a quenching group;

8. The reagent according to claim 7, characterized in that the target site of the specific complementary nucleic acid sequence is selected from the following group: wild-type PIK3CA H1047 site and mutant PIK3CA H1047R site.

9. The reagent according to claim 7, wherein the sequence of the specific complementary nucleic acid is selected from the group consisting of:

CACCATGATGTGCATCA, SEQ ID No:9;

CACCATGACGTGCATC, SEQ ID No: 10.

10. A kit, characterized in that the kit contains the reagent for detecting gene mutation according to claim 1.

11. Use of the reagent for detecting gene mutation according to claim 1 or the kit according to claim 10, characterized in that it is used to prepare a diagnostic product, wherein the diagnostic product is used to judge whether a subject is suitable for treatment with an EGFR targeted drug, or to pre-evaluate the effect of an EGFR targeted drug on a subject;

Wherein, the EGFR targeted drug is selected from the following group: gefitinib, erlotinib, icotinib, cetuximab, panitumumab, or a combination thereof.

12. The use according to claim 11, characterized in that the subject is an EGFR-positive tumor patient, and the tumor patient includes a leukemia patient.

13. The use according to claim 11, characterized in that the EGFR targeted drug is selected from the group consisting of erlotinib, icotinib, cetuximab, panitumumab, or a combination thereof.

14. An in vitro non-diagnostic method for detecting whether a sample contains a gene mutation, comprising the steps of:

(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, wherein the primer pair is selected from the following group:

Wherein, the reaction system further comprises:

(a1) a first probe used in conjunction with a first primer pair, wherein the first probe comprises: a probe shown in SEQ ID No: 3, a probe shown in SEQ ID No: 4, a probe shown in SEQ ID No: 5, and a probe shown in SEQ ID No: 6; and

(b1) a second probe used in conjunction with the second primer pair, wherein the second probe comprises: a probe shown in SEQ ID No: 9 and a probe shown in SEQ ID No: 10;

(S2) performing a PCR reaction on the PCR reaction system of step (S1) to obtain an amplified product;

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

15. The method according to claim 14, characterized in that the detection accuracy of the method is 0.06%-1%.

16. The method according to claim 14, characterized in that the detection accuracy of the method is 0.0625%-0.08%.