CN108949990A

CN108949990A - A kind of kit and method detecting EGFR genetic mutation

Info

Publication number: CN108949990A
Application number: CN201810896141.4A
Authority: CN
Inventors: 蒋析文; 刘悦; 朱小亚; 颜尔聪
Original assignee: Daan Gene Co Ltd Zhongshan University
Current assignee: Daan Gene Co Ltd Zhongshan University
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2018-12-07
Anticipated expiration: 2038-08-08
Also published as: CN108949990B

Abstract

The present invention provides a kind of kits and method for detecting EGFR genetic mutation, specifically, the method of EGFR genetic mutation, primer, probe and kit including the primed probe mixed liquor are detected the invention discloses a kind of, provide the primer and probe of detection C797S site mutation, detect the primer and probe of T790M site mutation, the primer and probe of 19del site mutation are detected, the primer and probe of L858R site mutation are detected.Using the present processes, 0.1% mutation rate can be detected based on digital pcr platform, had many advantages, such as that optimization process is simple, can examine more mutation type, absolute quantitation, high sensitivity, sample and easily obtained.

Description

A kind of kit and method detecting EGFR genetic mutation

Technical field

The invention belongs to biotechnologys and diagnosing tumor field, specifically, the present invention relates to detection non-small cell lung cancers The kit and method of EGFR genetic mutation.

Background technique

The morbidity and mortality of lung cancer occupy first place in the world, wherein and non-small cell lung cancer (NSCLC) accounts for 85% or more, And most of patient has been in cancer of late stage when making a definite diagnosis, the death rate is up to 80%-90%.Therefore, the treatment of advanced NSCLC Increasingly paid attention to by people.The double medicine combined chemotherapies of platiniferous are still the main means of advanced NSCLC treatment, but therapeutic effect is not It is very ideal.With the development of oncomolecularbiology technology, drug targeting therapeutic effect is more significant, opposite conventional cell poison Property drug, targeted therapy can act on tumor locus more accurately, can significantly reduce the damage to normal surrounding tissue, While reducing patient's adverse reaction rate, the accuracy for acting on tumour is improved.

EGF-R ELISA (EGFR) is a kind of transmembrane protein, is distributed widely on cell membrane, is receptor tyrosine One of kinase families member.The combination of EGFR and epidermal growth factor (EGF) can related gene in active cell core, to promote Cell division proliferation.EGFR is present in most cells, to the growth of tumour cell, breeding, transfer, new vascular generation, leaching Profit etc. plays an important role, and has expression in the kinds of tumors such as non-small cell lung cancer, colorectal cancer, breast cancer, prostate cancer.

In recent years, preferable using EGFR as effect of the molecular targeted therapy of target spot to advanced NSCLC patients.EGFR tyrosine Kinase inhibitor (EGFR-TKIs) is one of the critical treatment means of EGFR saltant type advanced NSCLC, in objective remission rate and nothing The double medicine scheme for combining of traditional platiniferous are significantly better than in terms of progress life cycle, but most of patient goes out after treatment 6-12 months Existing EGFR-TKIs drug resistance, resistance mechanism mainly includes primary drug resistance and acquired resistance.In NSCLC patient, EGFR is prominent It is flexible often to occur in 18-21 exon, wherein the 19th exons 1 9del site deletion and the 21st site exon L858R Mutation is the most common EGFR sensitizing mutation, accounts for about 90% be always mutated, is called classical mutation or hot spot mutation.20th exon T790M mutation be EGFR-TKI most common acquired resistance mutation, occur using EGFR-TKIs treatment NSCLC acquired About 50% detectable T790M mutation in drug resistant patient.Recently, EGFR20 exon C797S mutation is defined as the third generation The medicament-resistant mutation of TKI.For the treatment of NSCLC patient drug resistant for third generation TKI, with a kind of method of high sensitivity come really The mutation for determining the site C797S facilitates the state of an illness and finds ahead of time.Result of study confirms that in Chinese NSCLC patient, EGFR is always mutated Rate is about 30%, and wherein adenocarcinoma patients' mutation rate is about 50%, and women is non-, and adenocarcinoma patients may be up to 60%-70%.

Currently, tissue biopsy is the goldstandard of lesion detection, but since there are invasive drawn games to limit for the acquisition of tissue samples Property, most of advanced NSCLC patients or further consultation patient are difficult with the biopsy of repetition tissue to monitor body drug resistance situation and in real time The state of an illness, this allows for liquid Biopsy and comes into being.Liquid biopsy is mainly detection pair with blood free nucleic acid (cfDNA) As having many advantages, such as more fully to reflect tumor characteristic, overcoming Tumor Heterogeneity problem and Noninvasive that can repeatedly sample.Largely Studies have shown that cfDNA can be used as tissue it is undesirable when preferred substitution sample selected for assessing EGFR genetic mutation state Whether EGFR-TKI treatment method is used.CfDNA detection only needs a small amount of peripheral blood sample, it is easy to operate, noninvasive, have it is repeatable Property, it is continuously monitored suitable for being in progress to NSCLC conditions of patients, tumor prognosis evaluation, residual lesions detection etc..Due to cfDNA Concentration late in blood of patients with lung cancer is extremely low, therefore, more demanding to detection sensitivity, common detection in Gene Mutation Method is such as: Sanger PCR sequencing PCR, high-resolution melting point curve analytic approach, quantitative real-time PCR, denaturing high-performance chromatography skill Art etc. is difficult to meet its testing requirements.

Therefore, there is an urgent need in the art to develop effectively detect the technology of cfDNA in blood of patients with lung cancer and face to meet Bed demand.

Summary of the invention

The purpose of the present invention is to provide a kind of kits and method for detecting EGFR genetic mutation.

In the first aspect of the present invention, provide a kind of for digital pcr detection 20 exon genes of EGFR gene mutation Primer pair, the primer pair includes upstream primer and downstream primer；Wherein, the nucleotide sequence of the upstream primer such as SEQ Shown in ID NO.:16, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.:17.

The second aspect of the present invention provides a kind of probe for digital pcr detection 20 exons mutation of EGFR gene Group, the probe groups include nucleotide sequence the first mutant probe and/or nucleotide sequence as shown in SEQ ID NO.:18 The second mutant probe as shown in SEQ ID NO.:19.

In another preferred example, the probe groups further include nucleotide sequence wild spy as shown in SEQ ID NO.:20 Needle.

In another preferred example, 5 ' ends of the mutant probe include fluorescent reporter group FAM；And/or the mutation 3 ' ends of probe include fluorescent quenching group MGB.

In another preferred example, 5 ' ends of the wild probe include fluorescent reporter group VIC；And/or it is described wild 3 ' ends of probe include fluorescent quenching group MGB.

The third aspect of the present invention provides a kind of kit for digital pcr detection EGFR genetic mutation, the examination Agent box includes the first primer pair, and the first primer is for primer pair described in first aspect present invention.

In another preferred example, the kit further includes the first probe groups, and first probe groups are the present invention second Probe groups described in aspect.

In another preferred example, the kit further includes the second primer pair, and second primer pair includes SEQ ID Downstream primer shown in upstream primer shown in NO.:8 and SEQ ID NO.:9.

In another preferred example, the kit further includes third primer pair, and the third primer pair includes SEQ ID Downstream primer shown in upstream primer shown in NO.:1 and SEQ ID NO.:2.

In another preferred example, the kit further includes the 4th primer pair, and the 4th primer pair includes SEQ ID Downstream primer shown in upstream primer shown in NO.:12 and SEQ ID NO.:13.

In another preferred example, the kit further includes the 5th primer pair, and the 5th primer pair includes SEQ ID Downstream primer shown in upstream primer shown in NO.:5 and SEQ ID NO.:6.

In another preferred example, the kit further includes the second probe groups, and second probe groups include SEQ ID Mutant probe shown in NO.:10；And/or wild probe shown in SEQ ID NO.:11.

In another preferred example, the kit further includes third probe groups, and the third probe groups include SEQ ID PCR amplification blocking agent shown in NO.:3, open country shown in mutant probe and/or SEQ ID NO.:7 shown in SEQ ID NO.:4 Raw probe.

In another preferred example, the kit further includes the 4th probe groups, and the 4th probe groups include SEQ ID Mutant probe shown in NO.:14；And/or wild probe shown in SEQ ID NO.:15.

In another preferred example, the kit includes the first container, includes the first primer in the first container To and first probe groups.The first container is arranged for detection 20 exon C797S site mutation of EGFR gene.

In another preferred example, the kit further includes second container, is drawn in the second container comprising described second Object to and second probe groups.It is prominent that the second container is arranged for detection 20 site exon T790M of EGFR gene Become.

In another preferred example, the kit further includes third container, is drawn in the third container comprising the third Object is to, five primer pair and the third probe groups.The third container is arranged for detection 19 exon of EGFR gene 19del deletion segment.

In another preferred example, the kit further includes the 4th container, is drawn in the 4th container comprising the described 4th Object to and the 4th probe groups.It is prominent that 4th container is arranged for detection 21 site exon L858R of EGFR gene Become.

In another preferred example, the kit further includes positive control and/or negative control.

In another preferred example, the kit further includes digital pcr reaction enzymes；And/or digital pcr buffer.

In another preferred example, the ultimate density of each upstream primer and each downstream primer in the reaction system is 0.1~10 μ mol/L；More preferably 0.5~5 μm of ol/L.

In another preferred example, each mutant probe final concentration in the reaction system and each wild probe be in the reaction system Final concentration ratio be 2:1.

In another preferred example, final concentration of 0.1~5 μm of ol/L of each mutant probe in the reaction system；Preferably 0.1 ~1 μm of ol/L.

In another preferred example, final concentration of 0.05~2 μm of ol/L of each wild probe in the reaction system；Preferably 0.1~0.5 μm of ol/L.

The fourth aspect of the present invention provides a kind of method of digital pcr detection EGFR genetic mutation, the method includes Step:

(1) DNA sample of object to be detected is provided；

(2) it prepares digital pcr reaction system and carries out digital pcr detection:

Wherein, the digital pcr reaction system includes the first digital pcr reaction system, the first digital pcr reactant System includes the DNA sample, primer pair and the second aspect of the present invention institute described in first aspect present invention that step (1) provides The probe groups stated.It is prominent that the first digital pcr reaction system is arranged for detection 20 site exon C797S of EGFR gene Become.

In another preferred example, the digital pcr reaction system further includes the second digital pcr reaction system, and described second Digital pcr reaction system includes the DNA sample, second primer pair and second probe groups that step (1) provides. The second digital pcr reaction system is arranged for detection 20 exon T790M site mutation of EGFR gene.

In another preferred example, the digital pcr reaction system further includes third digital pcr reaction system, the third Digital pcr reaction system includes the DNA sample, the third primer pair, five primer pair and described that step (1) provides Third probe groups.The third digital pcr reaction system is arranged for detection 19 exons 1 9del of EGFR gene missing position Point.

In another preferred example, the digital pcr reaction system further includes the 4th digital pcr reaction system, and the described 4th Digital pcr reaction system includes the DNA sample, the 4th primer pair and the 4th probe groups that step (1) provides. The 4th digital pcr reaction system is arranged for detection 21 exon L858R site mutation of EGFR gene.

In another preferred example, the first digital pcr reaction system, the second digital pcr reaction system, described Three digital pcr reaction systems and the 4th digital pcr reaction system independently exist.

In another preferred example, the DNA sample carrys out autoblood free nucleic acid.

In another preferred example, the method is the detection method of non-diagnostic purpose.

In another preferred example, the digital pcr reaction system further includes positive control and/or negative control.

In another preferred example, the digital pcr reaction system further includes digital pcr reaction enzymes；And/or digital pcr is used Buffer.

In another preferred example, the ultimate density of upstream primer and downstream primer in the reaction system is 0.1~10 μm of ol/ L；More preferably 0.5~5 μm of ol/L.

In another preferred example, the end of final concentration and wild probe in the reaction system of mutant probe in the reaction system Concentration ratio is 2:1.

In another preferred example, final concentration of 0.1~5 μm of ol/L of the mutant probe in the reaction system；Preferably 0.1~1 μm of ol/L.

In another preferred example, final concentration of 0.05~2 μm of ol/L of the wild probe in the reaction system；Preferably 0.1~0.5 μm of ol/L.

The fifth aspect of the present invention provides primer pair described in first aspect present invention, and/or second aspect of the present invention The purposes of the probe groups is used to prepare digital pcr detection kit, and the digital pcr detection kit is for detecting 20 exons mutation of EGFR gene.

In another preferred example, it is described sport the 20th exon of EGFR gene c.2389T > A mutation and/or c.2390G > C mutation.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1: the dPCR result schematic diagram of the present invention detection mutational site C797S negative control；

Fig. 2: the present invention detects the dPCR result schematic diagram of the mutational site C797S gDNA control；

Fig. 3: the dPCR result schematic diagram of the present invention detection mutational site C797S positive control；

Fig. 4: the dPCR result schematic diagram of the present invention detection mutational site C797S sample DNA template；

Fig. 5: the testing result of control primer pair 1 is used；

Fig. 6: the testing result of control primer pair 2 is used；

Fig. 7: the testing result of control probe 1 is used；

Fig. 8: the testing result of control probe 2 is used.

Specific embodiment

The present inventor obtains a kind of detection EGFR genetic mutation especially the 20th exon by extensive and in-depth research C797S is mutated the kit and method of (c.2389T > A and c.2390G > C), the experimental results showed that, method of the invention and reagent Box can detect 0.1% mutation rate based on digital pcr platform, have that detection process is simple, it is more to examine mutation type, absolutely calmly The advantages that amount, high sensitivity, sample easily obtain.

The present invention relates to non-small cell lung cancer detection technique fields, disclose a kind of detection Patients with Non-small-cell Lung blood Method, primer, probe and the kit including the primed probe mixed liquor of free nucleic acid EGFR genetic mutation.Provide detection The primer and probe of C797S site mutation, detect the primer and probe of 19del site mutation, and detection T790M site mutation draws Object and probe detect the primer and probe of L858R site mutation.The application is with Patients with Non-small-cell Lung blood free nucleic acid Template can also directly acquire mutant proportion while measuring EGFR genetic mutation.This kit is prominent to the C797S of EGFR gene Become, T790M mutation, 19del mutation, L858R mutation four 22 seed types of site point, 4 pipes detection, can be examined based on digital pcr platform 0.1% mutation rate out has optimization process is simple, can examine more mutation type, absolute quantitation, high sensitivity, sample easily to obtain The advantages that.

Specifically the present invention provides one kind to be based on digital pcr platform, detects Patients with Non-small-cell Lung EGFR base Because of the method for mutation, primer, probe and kit including the primed probe mixed liquor, detection sample is that non-small cell lung cancer is suffered from The blood free nucleic acid of person.

Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.

Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated " about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes 101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).

Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method And material, place enumerates preferred method and material herein.

Digital pcr (dPCR)

Digital pcr (dPCR) is able to achieve the absolute quantitation of nucleic acid molecules, has when detecting the EGFR genetic mutation of cfDNA Higher sensitivity and specificity.It is assigned to up to ten thousand by way of physically or chemically dividing, by a PCR reaction system In small reactor, includes in each microreactor or not comprising the target nucleic acid molecules that one or more are copied, carry out " unimolecule template PCR amplifications ".After amplification, original sample is calculated by the number and statistical method of positive reaction unit The copy number of middle target gene.Therefore, dPCR can be directly determined down to single without establishing standard curve and be copied the exhausted of target molecule to be checked To number.But requirement of the digital pcr to primer and probe is all very high, a little change of primer and probe sequence just will affect The specificity and sensitivity of detection.

A large number of studies show that EGFR genetic mutation and TKI curative effect are closely related.Therefore, go out as EGFR-TKIs is drug resistant It is existing, at the same detect EGFR gene C797S site mutation in blood free nucleic acid, T790M site mutation, 19del site deletion and L858R site mutation can provide reference for the screening of patients with lung cancer targeted therapy, and can be resistance to during real-time monitoring patient medication Medicine mutation.To sum up, it needs a kind of fast and reliable ground while 20 exon C797S of EGFR gene can be detected in high sensitivity at this stage Mutation, 20 exon T790M mutation, 19 exons 1 9del missing and 21 exon L858R be mutated the mutation of 22 kinds of four sites The method and kit of type.

The present invention provides a kind of methods for detecting NSCLC blood samples of patients free nucleic acid EGFR genetic mutation, primer, probe And its kit, have that optimization process is simple, can examine more mutation type, absolute quantitation, high sensitivity, sample and easily obtain etc. and is excellent Point.

Technical scheme is as follows:

It is a kind of to detect the method for NSCLC blood samples of patients free nucleic acid EGFR genetic mutation, primer, probe and including the primer The kit of probe mixed liquor.It provides for specific primer and probe needed for detecting 20 mutational sites exon C797S, For specific primer and probe needed for detecting 20 mutational sites exon T790M, for detecting 19 exons 1 9del missing Specific primer needed for site and probe, for specific primer and spy needed for detecting 21 mutational sites exon L858R Needle.The application can also directly acquire prominent using NSCLC blood samples of patients free nucleic acid as template while measuring EGFR genetic mutation Control with changed scale.This kit is mutated 20 exon C797S of EGFR gene, 20 exon T790M are mutated, 19 exons 1 9del are lacked It loses and 21 exon L858R is mutated four 22 seed types of site and is detected, 0.1% mutation can be detected based on dPCR platform Rate, has many advantages, such as can to examine that mutation type is more, optimization process is simple, absolute quantitation, high sensitivity, sample easily obtain；

It is preferably carried out in mode at of the invention one, it is free that the invention discloses a kind of detection NSCLC blood samples of patients Primer, the probe of nucleic acid EGFR genetic mutation:

The probe includes the mutant probe and internal control probe for detecting the site 19del, detects the mutant probe in the site T790M With wild probe, the mutant probe and wild probe in the site L858R are detected；5 ' ends of the mutant probe are marked with FAM fluorescence 5 ' ends of reporter group, the internal control probe and wild probe are marked with VIC fluorescent reporter group, the mutant probe, internal control 3 ' ends of probe and wild probe are marked with MGB fluorescent quenching group；

It is described detection 19 exons 1 9del site mutations upstream primer nucleotide sequence as shown in SEQ ID NO:1, under The nucleotide sequence of primer is swum as shown in SEQ ID NO:2, the nucleotide sequence of PCR amplification blocking agent such as SEQ ID NO:3 institute Show, the nucleotide sequence of mutant probe is as shown in SEQ ID NO:4；Also add in the primer and probe for detecting 19del Added with the primer and probe as internal control, the upstream primer nucleotide sequence as internal control as shown in SEQ ID NO:5, internal control Downstream primer nucleotide sequence is as shown in SEQ ID NO:6, and the probe nucleotide sequence of internal control is as shown in SEQ ID NO:7；Institute 3 ' the ends for stating SEQ ID NO:3 nucleotide sequence are marked with ZIP, and 5 ' ends of the SEQ ID NO:4 nucleotide sequence are marked with FAM, 3 ' ends are marked with MGB, and 5 ' ends of the SEQ ID NO:7 nucleotide sequence are marked with VIC, 3 ' ends are marked with MGB；

The PCR amplification blocking agent is in conjunction with the wild-type sequence in 19 site exons 1 9del of EGFR gene, specificity resistance Disconnected amplification of the upstream and downstream primer to target fragment, mutant probe do not discharge fluorescence signal；When 19del site mutation, mutant probe In conjunction with the target fragment that upstream and downstream primer is expanded, FAM fluorescence signal is discharged；The internal control primer and probe are according to people EGFR gene conservative fragments design and synthesize, prominent for detecting c.2235~2257 whole that 19 exon of EGFR gene occurs Become；

It is described detection 20 exon T790M site mutations upstream primer nucleotide sequence as shown in SEQ ID NO:8, The nucleotide sequence of downstream primer as shown in SEQ ID NO:9, the nucleotide sequence of mutant probe as shown in SEQ ID NO:10, The nucleotide sequence of wild probe is as shown in SEQ ID NO:11；5 ' ends of the SEQ ID NO:10 nucleotide sequence are marked with FAM, 3 ' ends are marked with MGB, and 5 ' ends of the SEQ ID NO:11 nucleotide sequence are marked with VIC, 3 ' ends are marked with MGB；

The nucleotide sequence such as SEQ ID NO:12 institute of the upstream primer of 21 exon L858R site mutations of the detection Show, the nucleotide sequence of downstream primer is as shown in SEQ ID NO:13, the nucleotide sequence of mutant probe such as SEQ ID NO:14 Shown, the nucleotide sequence of wild probe is as shown in SEQ ID NO:15；5 ' ends of the SEQ ID NO:14 nucleotide sequence Be marked with FAM, 3 ' ends are marked with MGB, 5 ' ends of the SEQ ID NO:15 nucleotide sequence are marked with VIC, 3 ' ends are marked with MGB。

The nucleotide sequence such as SEQ ID NO:16 institute of the upstream primer of 20 exon C797S site mutations of the detection Show, the nucleotide sequence of downstream primer is as shown in SEQ ID NO:17, the nucleotide sequence of mutant probe 1 such as SEQ ID NO:18 Shown, the nucleotide sequence of mutant probe 2 is as shown in SEQ ID NO:19, the nucleotide sequence of wild probe such as SEQ ID NO: Shown in 20；5 ' ends of the SEQ ID NO:18 nucleotide sequence are marked with FAM, 3 ' ends are marked with MGB, the SEQ ID NO: 5 ' ends of 19 nucleotide sequences are marked with FAM, 3 ' ends are marked with MGB, 5 ' end marks of the SEQ ID NO:20 nucleotide sequence Note has VIC, 3 ' ends to be marked with MGB.

Preferably, the final concentration of 1.0 μm of ol/L of the upstream primer in the reaction system, the downstream primer are being reacted Final concentration of 1.0 μm of ol/L in system, the final concentration of 0.25 μm of ol/L of the mutant probe in the reaction system, the open country The final concentration of 0.25 μm of ol/L of raw probe in the reaction system, the internal control probe in the reaction system final concentration of 0.125μmol/L；

The primer probe sequence in each mutational site is as follows,

Detect the primed probe nucleotide sequence information of 20 exon C797S site mutations:

Detect the primed probe nucleotide sequence information of 20 exon T790M site mutations:

Detect the primed probe nucleotide sequence information of 19 exons 1 9del deletion segments:

Detect the primed probe nucleotide sequence information of 21 exon L858R site mutations:

Above-mentioned PCR primer and the probe for indicating different type fluorescence can be used for carrying out wild type and mutant DNA template Detection, according to the different fluorescence that result is presented, judgement sample DNA profiling type；Above-mentioned primer and probe is according to Human epidermal growth factor receptor gene 19th exon, 20 exons, 21 exon difference mutational sites gene order design and synthesize, can detecte EGFR gene 19 exons, 20 exons, 21 exons 22 kinds of four sites mutation type；The common mutations type of EGFR gene is as follows,

The 20th site exon C797S of EGFR gene c.2389T > (mutation type can join for A and c.2390G > C Substitution Examine document Song H N, Jung K S, Yoo K H, et al.Acquired C797S Mutation upon Treatment with a T790M-Specific Third-Generation EGFR Inhibitor(HM61713)in Non-Small Cell Lung Cancer.[J].Journal of Thoracic Oncology,2016,11(4):e45-e47.)；

The 20th site exon T790M of EGFR gene c.2369C > (mutation type can refer to document to T Substitution Takahama T,Sakai K,Takeda M,et al.Detection of the T790M mutation of EGFR in plasma of advanced non–small cell lung cancer patients with acquired resistance to tyrosine kinase inhibitors(West Japan oncology group 8014LTR study)[J].Oncotarget,2016,7(36):58492-58499.)；

The 19th exons 1 9del of EGFR gene mutation occur nucleotide sequence c.2235~2257 between any missing (specific mutation type can refer to document Wei W, Jilin L I, Ning S, et al.Comparison of to mutation type prognosis between EGFR 19-del and L858R mutations in patients with non-small- cell lung cancer[J].Shandong Medical Journal,2016.)；

The 21st site exon L858R of EGFR gene c.2573T > G Substitution (mutation type can refer to document Wei W, Jilin L I,Ning S,et al.Comparison of prognosis between EGFR 19-del and L858R mutations in patients with non-small-cell lung cancer[J].Shandong Medical Journal,2016.)；

The specific primer and probe can accurately detect the EGFR genetic mutation class using blood free nucleic acid as template Type can immediately arrive at the frequency of mutation of gene loci while detecting mutation.Using mutant probe and internal control probe in detecting 19 exon genes deletion segments, are determined by copy number, detect the ratio of different fluorescence signals and its copy number to determine 19 Whether exon occurs gene mutation and the ratio of gene mutation occurs.Using mutant probe and wild 20 exon of probe in detecting With the mutated gene of 21 exons, when examined gene mutates, the nucleotide piece of mutant probe and upstream and downstream primer amplification Section combines；When examined gene does not mutate, wild probe is in conjunction with the nucleotide fragments that upstream and downstream primer expands；Pass through inspection The copy number of different fluorescence signals and jump reaction and the ratio of total copy number (mutant copies number+wild copy number) are surveyed, is come true Whether fixed examined site has occurred gene mutation and the ratio of gene mutation occurs, and present invention combination dPCR system can detect 0.1% mutant proportion.

Kit prepared by above-mentioned specific primer and probe can detect four sites of EGFR gene based on dPCR platform 22 kinds of mutation types provide reference for whether patient needs to carry out targeted therapy, it can also be used to which the high sensitivity of NSCLC patient is early The real-time monitoring of resistance mutation during phase detection and medication.

It is preferably carried out in mode in of the invention another, the invention also discloses a kind of detection NSCLC blood samples of patients The kit of free nucleic acid EGFR genetic mutation, including for preparing dPCR reaction solution primed probe mixed liquor, Mastermix, Solution, check sample, sterile water and sealing fluid, wherein primed probe mixed liquor includes following ingredient, such as Table 1,

1 primed probe mixed liquor of table

Wherein, described prominent for detecting the mutational site C797S, the mutational site T790M, the mutational site 19del and L858R Primer and the probe difference for conjugating point are as follows:

The nucleotide sequence of the upstream primer of 20 exon C797S site mutations is detected as shown in SEQ ID NO:16, under Swim primer nucleotide sequence as shown in SEQ ID NO:17, the nucleotide sequence of mutant probe 1 as shown in SEQ ID NO:18, The nucleotide sequence of mutant probe 2 is as shown in SEQ ID NO:19, the nucleotide sequence of wild probe such as SEQ ID NO:20 institute Show；5 ' ends of the SEQ ID NO:18 nucleotide sequence are marked with FAM, 3 ' ends are marked with MGB, the SEQ ID NO:19 core 5 ' ends of nucleotide sequence are marked with FAM, 3 ' ends are marked with MGB, and 5 ' ends of the SEQ ID NO:20 nucleotide sequence are marked with VIC, 3 ' ends are marked with MGB；

Detect the primed probe of 20 exon T790M site mutations, including the upstream primer core as shown in SEQ ID NO:8 Nucleotide sequence, the downstream primer nucleotide sequence as shown in SEQ ID NO:9, the mutant probe core as shown in SEQ ID NO:10 Nucleotide sequence, the wild probe nucleotide sequence as shown in SEQ ID NO:11；The SEQ ID NO:10 nucleotide sequence 5 ' ends are marked with FAM, 3 ' ends are marked with MGB, and 5 ' ends of the SEQ ID NO:11 nucleotide sequence are marked with VIC, 3 ' end marks Note has MGB；

The probe includes the mutant probe and internal control probe for detecting the site 19del, detects the mutant probe in the site T790M With wild probe, the mutant probe and wild probe in the site L858R are detected, detects the mutant probe 1 in the site C797S, mutation is visited Needle 2 and wild probe；5 ' ends of the mutant probe are marked with FAM fluorescent reporter group, the internal control probe and wild probe 5 ' ends be marked with VIC fluorescent reporter group, it is glimmering that 3 ' ends of the mutant probe, internal control probe and wild probe are marked with MGB Optical quenching group；

Detect the primed probe of 19 exons 1 9del site mutations, including the upstream primer core as shown in SEQ ID NO:1 Nucleotide sequence, the downstream primer nucleotide sequence as shown in SEQ ID NO:2, the PCR amplification as shown in SEQ ID NO:3 block Agent nucleotide sequence, the mutant probe nucleotide sequence as shown in SEQ ID NO:4；It detects in the primer and probe of 19del also Added with the primer and probe as internal control, including the upstream primer nucleotide sequence by the be shown as internal control of SEQ ID NO:5, By the downstream primer nucleotide sequence of the be shown as internal control of SEQ ID NO:6, by the be shown as internal control probe of SEQ ID NO:7 Nucleotide sequence；3 ' ends of the SEQ ID NO:3 nucleotide sequence are marked with ZIP, the SEQ ID NO:4 nucleotide sequence 5 ' ends be marked with FAM, 3 ' ends are marked with MGB, 5 ' ends of the SEQ ID NO:7 nucleotide sequence are marked with VIC, 3 ' end marks Note has MGB；

The PCR amplification blocking agent is in conjunction with the wild-type sequence in 19 site exons 1 9del of EGFR gene, specificity resistance Disconnected amplification of the upstream and downstream primer to target fragment, mutant probe do not discharge fluorescence signal；When 19del site mutation, mutant probe In conjunction with the target fragment that upstream and downstream primer is expanded, FAM fluorescence signal is discharged；The internal control primer and probe are according to people EGFR gene conservative fragments design and synthesize, for detecting whole EGFR genes containing 19 exons；

Detect the primed probe of 21 exon L858R site mutations, including the upstream primer as shown in SEQ ID NO:12 Nucleotide sequence, the downstream primer nucleotide sequence as shown in SEQ ID NO:13 are mutated as shown in SEQ ID NO:14 and visit Needle nucleotide sequence, the wild probe nucleotide sequence as shown in SEQ ID NO:15；The SEQ ID NO:14 nucleotides sequence 5 ' ends of column are marked with FAM, 3 ' ends are marked with MGB, and 5 ' ends of the SEQ ID NO:15 nucleotide sequence are marked with VIC, 3 ' End is marked with MGB；

Preferably, the final concentration of 1.0 μm of ol/L of the upstream primer in the reaction system, the downstream primer are being reacted Final concentration of 1.0 μm of ol/L in system, the final concentration of 0.25 μm of ol/L of the mutant probe in the reaction system, the open country The final concentration of 0.125 μm of ol/L of raw probe in the reaction system, the internal control probe in the reaction system final concentration of 0.125μmol/L；

The Mastermix of the dPCR reaction solution includes following ingredient, such as table 2,

2 Mastermix of table

Number	Component	Main component in component
			1	Mastermix	dNTPs、KCl、Tris-HCl、MgCl₂, DTT, Taq enzyme

The reference substance includes following ingredient, such as table 3,

3 check sample of table

Number	Component	Main component in component
			1	Negative control	Axenic purification water
2	GDNA control	Cell strain DNA mixed liquor
			3	Positive control	Cell strain DNA mixed liquor, outer conjunction large fragment mixed liquor

Preferably, contain Caco2 cell strain nucleic acid in the cell strain DNA mixed liquor of the gDNA control；

Contain in the cell strain DNA mixed liquor of the positive control:

Detect outer conjunction large fragment 1, the outer conjunction large fragment 2 of 20 exon C797S mutation；

Detect the H1975 cell strain DNA of 20 exon T790M mutation；

Detect the H1650 cell strain DNA of 19 exons 1 9del deletion segments；

Detect the H1975 cell strain DNA in 21 mutational sites exon L858R.

It is blood free nucleic acid that kit of the present invention, which is applicable in sample,.

Kit of the present invention be used for determines detect validity standard are as follows: every time detect be respectively provided with negative control group, GDNA control group and positive controls, when the positive controls of testing result are the positive, and negative control group and gDNA control group When being feminine gender, show that experimental result is effective.The detection sensitivity of kit of the present invention can reach 0.1%.

It is preferably carried out in mode in of the invention another, the invention also discloses a kind of detection NSCLC blood samples of patients The method of free nucleic acid EGFR genetic mutation, specific steps include:

1. processing sample to be tested simultaneously extracts sample DNA template；Preferably, sample to be tested is blood free nucleic acid；

2. prepare dPCR reaction system, by sample to be tested DNA profiling, specific primer and probe, Mastermix, Solution and sterile water mixing, are prepared dPCR reaction solution；DPCR reaction solution constitutes such as table 4,

4 dPCR reaction solution of table

Specific primer and probe	1μL
		DNA profiling	5μL
Mastermix	7.5μL
		Solution	0.75μL
Sterile water	Complement to 16 μ L

Preferably, the primed probe mixed liquor be it is above-mentioned for detect 19del deletion segment sequence SEQ ID NO:1~ The primer and probe of SEQ ID NO:7, or detect drawing for the mutational site T790M sequence SEQ ID NO:8~SEQ ID NO:11 Object and probe, or the primer and probe of the detection mutational site L858R sequence SEQ ID NO:12~SEQ ID NO:15, or detection One or more of primer and probe of the mutational site C797S sequence SEQ ID NO:16~SEQ ID NO:20；

It is highly preferred that the final concentration of 1.0 μm of ol/L of the upstream primer in the reaction system, the downstream primer is anti- Answer final concentration of 1.0 μm of ol/L in system, the final concentration of 0.25 μm of ol/L of the mutant probe in the reaction system is described The final concentration of 0.125 μm of ol/L of wild probe in the reaction system, the internal control probe in the reaction system final concentration of 0.125μmol/L；

3. the dPCR reaction solution for taking 15 μ L to prepare, by Clarity^TMAutomatic sample adding instrument is automatic by it, is equably subdivided into The reaction member of 10 000 nanoliter levels；Using Clarity^TMSealing instrument is added sealing fluid and is sealed to reaction member.

4. carrying out PCR amplification in dedicated PCR instrument, the amplification condition of the PCR are as follows: first stage, 95 DEG C of initial denaturations 10 Minute；Second stage, 94 DEG C are denaturalized 30 seconds, and 56 DEG C extend 30 seconds, 40 circulations；Phase III, 98 DEG C are stablized micro- 10 points of reaction Clock；Final 4 DEG C of terminations reaction；

5. using Clarity^TMSystem software analyzes testing result, and is calculated respectively based on Poisson distribution Statistics The copy number of target molecules in sample；

6. determining that sample to be tested whether there is mutation, and obtain mutant proportion according to the intensity and ratio of each fluorescence signal.

The application is as follows using dPCR testing principle: dPCR technology is that the reaction reagent in single PCR pipe is divided into about Micro- reactions up to ten thousand in each micro- reaction or are free of nucleic acid target molecule to be checked, or contain 1 to several separate target nucleic acids to be checked Son, and each micro- reaction is used as an independent PCR reaction member.After PCR process, fluorescence is carried out one by one to micro- reaction Detection identifies that Yin/Yang is reacted.Micro- reaction containing different DNA profilings releases different fluorescence signals, and template is not micro- Reaction does not generate fluorescence signal then.Finally, calculating target to be checked according to the ratio of Poisson distribution Statistics and positive micro- reaction Mark molecule copy number.Since the interpretation of dPCR result only determines whether amplification, Ct value is not depended on, to the resistance to of PCR reaction suppressor Greatly improved by ability, without necessarily referring to product and standard curve can accurate quantification, provided for this patent a kind of completely new Technical thought and means.

Detection method

The present invention proposes a kind of method, primer, probe and reagent for detecting Patients with Non-small-cell Lung EGFR genetic mutation Box, the kit are based on dPCR platform, can detect the mutation rate of mutated gene 0.1%.Specific implementation step is as follows,

Step 1: sample to be tested DNA profiling extracts

It extracts 2~10mL of NSCLC Venous Blood to be placed in noninvasive heparin tube, marking ensures that label information is errorless Afterwards, it is stored at room temperature, blood plasma is isolated in 12 hours, is preferably immediately disconnected blood plasma, the extraction for sample DNA.Mountain in use The blood free nucleic acid extracts kit of Da An gene limited liability company of university, kit article No. be cat.#DA0670 or Cat.#DA0680 is carried out according to kit specification the extraction of free nucleic acid；Institute is measured using 2.0 fluorescent quantitation instrument of Qubit Propose the purity and concentration of nucleic acid, template DNA can be directly used for subsequent experimental, or be placed in -20 DEG C of refrigerators freeze it is spare；

Step 2: the preparation of dPCR system

DPCR system prepare before prepare: take out kit in specific primer and probe, Mastermix, Solution, Sterile water etc., room temperature are melted, and the concussion that is vortexed is centrifuged 10 seconds after mixing, and prepare dPCR system；DPCR system constitutes such as table 5:

5 dPCR system of table

Specific primer and probe	1μL
		Mastermix	7.5μL
Solution	0.75μL
		Sterile water	Complement to 11 μ L

The nucleotide sequence information difference of the primed probe is as follows:

The probe includes the mutant probe and internal control probe for detecting the site 19del, detects the mutant probe in the site T790M With wild probe, the mutant probe and wild probe in the site L858R, the mutant probe in the detection site C797S and wild spy are detected Needle；The 5 ' of the mutant probe, which are held, is marked with FAM fluorescent reporter group, and 5 ' ends of the internal control probe and wild probe are marked with 3 ' ends of VIC fluorescent reporter group, the mutant probe, internal control probe and wild probe are marked with MGB fluorescent quenching group；

Step 3: sample-adding

Take the 5 μ L of each check sample in 5 μ L of sample DNA template and kit prepared by step 1, sample-adding to step 2 In eight connecting legs of the dPCR reaction system of preparation, make the 16 μ L of total volume of every pipe dPCR reaction solution；Eight connecting leg pipe lids are covered tightly, are filled Divide and mix, high speed centrifugation 10 seconds, is used to prepare micro- reaction；The check sample of the kit such as table 6:

The check sample of 6 kit of table

Number	Component	Main component in component
			1	Negative control	Axenic purification water
2	GDNA control	Caco2 cell strain DNA
			3	Positive control	Cell strain DNA mixed liquor, outer conjunction large fragment mixed liquor

The gDNA check sample is 19del, T790M, L858R and C797S wild-type nucleic acid sample containing EGFR gene This, derives from Caco2 cell strain DNA；Positive control is that wild type Caco2 cell strain DNA and 4 kinds are contained saltant type template DNA is mixed in a certain ratio respectively；Wherein, the DNA profiling containing the mutational site the EGFR gene 19del positive derives from H1650 Cell strain, the DNA profiling containing the mutational site the EGFR gene T790M positive derive from H1975 cell strain, contain EGFR gene The positive DNA profiling in the mutational site L858R derives from H1975 cell strain, positive containing the mutational site EGFR gene C797S DNA profiling derives from outer conjunction large fragment 1, outer conjunction large fragment 2；Negative control sample is axenic purification water；

Step 4: preparing micro- reaction

The dPCR reaction solution for taking 15 μ L to prepare, by Clarity^TMAutomatic sample adding instrument is automatic by it, is equably subdivided into 10 The reaction member of 000 nanoliter level；Using Clarity^TMSealing instrument is added sealing fluid and is sealed to reaction member；

Step 5: PCR amplification

96 orifice plates after sealer are put into PCR instrument, are expanded, the reaction condition of PCR amplification: the first stage, 95 DEG C pre- Denaturation 10 minutes；Second stage, 94 DEG C are denaturalized 30 seconds, and 56 DEG C extend 30 seconds, 40 circulations；Phase III, 98 DEG C stablize it is micro- anti- It answers 10 minutes；Final 4 DEG C of terminations reaction；

Step 6: result is read and analysis

PCR product is put into Clarity^TMIn reading apparatus, software is opened, completes the setting of detection sample essential information；Inspection After the completion of survey, the value in the channel FAM and VIC channel fluorescence signal is checked；By negative control sample, positive control and without mould The reference of plate check sample and the distribution of fluorescence signal, are adjusted micro- reaction signal threshold value.

Main advantages of the present invention are:

(1) provided by the present invention for the 20th mutational site exon C797S position purpose core of augmentation detection EGFR gene The primer pair of acid fragment, sensitivity is high, can effectively be expanded to plasma free nucleic acid, can satisfy digital pcr completely Needs；

(2) it present invention optimizes specific mutations probe, obtains for the 20th site exon C797S mesh of EGFR gene The high mutant probe of nucleic acid fragment specificity, thus significant increase Detection accuracy.

(3) the present invention also provides EGFR gene C797S in a kind of detection NSCLC patients blood plasma's free nucleic acid to be mutated, T790M is mutated, 19del is mutated, method, primer, probe and the kit of L858R mutation four sites, 22 kinds of mutation types.This Kit optimizes specific primer probe, for a kind of mutational site only need the probe of pair of primers and a set of fluorescent marker into Row detection only needs 4 pipes to can be detected 22 kinds of mutation types of EGFR using specific primer provided by the invention and probe, can EGFR genetic mutation rate is calculated, can detect 0.1% mutant proportion, high sensitivity.

(4) mutation type is more, process optimization is simple, required sample is few, performance is steady with that can examine for detection method of the invention It is fixed efficiently, high accuracy the advantages that, the difference individually copied can be told, realize absolute quantitation truly, data point Analysis automation, as a result can observe in real time.

(5) detection method of the invention, the complete stopped pipe operation of detection process, effectively reduces the risk of PCR product pollution. Method of the invention is suitable for Patients with Non-small-cell Lung EGFR-TKI resistance mechanism and targets the assessment of medication, realizes treatment The dynamic tracing of effect and real-time monitoring to patient's prognosis, be explore non-small cell lung cancer early diagnosis with efficiently treat one Feasible way.

The present invention is suitable for Patients with Non-small-cell Lung EGFR-TKI resistance mechanism and targets the assessment of medication, and realization is controlled The dynamic tracing of therapeutic effect and real-time monitoring to patient's prognosis are explored non-small cell lung cancer early diagnosis and are efficiently treated One feasible way, is worthy of popularization.In addition, method of the invention is equally applicable to non-diagnostic purpose, for example, in new drug In R&D process, the gene mutation information for being used as intermediate result, these gene mutations letter are obtained using detection method of the invention Breath can be used as the need of public health management, can be used for research and the targeting new drug development of EGFR-TKI resistance mechanism.

Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002) Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.

Embodiment 1

Present embodiments provide it is a kind of detect Patients with Non-small-cell Lung EGFR genetic mutation kit constituent, Packaging and quantity (48 reactions/box), such as table 7,

Constituent, packaging and the quantity of 7 kit of table

Embodiment 2: sensitivity technique and mutation recall rate experiment

GDNA check sample is the nucleic acid containing EGFR gene 19del wild type, the nucleic acid of T790M wild type, the open country L858R The nucleic acid of raw type and the sample of nucleic acid of C797S wild type, derive from Caco2 cell strain DNA；Sensitivity reference material is by wild type Caco2 cell strain (being purchased from Shanghai Inst. of Life Science, CAS) DNA and 4 kinds of saltant type template DNAs are pressed centainly respectively Ratio mixing, the mutation rate of mixed liquor is respectively 10%, 5%, 1%, 0.1%；Wherein, position is mutated containing EGFR gene 19del The DNA of point derives from H1650 cell strain, and the DNA containing the mutational site EGFR gene T790M (is purchased from from H1975 cell strain Shanghai Inst. of Life Science, CAS), the DNA containing the mutational site EGFR gene L858R derives from H1975 cell Strain, the DNA containing the mutational site EGFR gene C797S derive from artificial synthesized large fragment 1,2；Negative control is sterile water；

Negative control, gDNA control, each 5 μ L of sensitivity reference material are taken, the dPCR reaction system prepared to step 2 is loaded Eight connecting legs in, make every 16 μ L of pipe dPCR reaction solution total volume；Eight connecting leg pipe lids are covered tightly, are mixed well, high speed centrifugation 10 seconds, It is prepared for micro- reaction；

The dPCR reaction solution for taking 15 μ L to prepare, by Clarity^TMAutomatic sample adding instrument is automatic by it, is equably subdivided into 10 The reaction member of 000 nanoliter level；Using Clarity^TMSealing instrument is added sealing fluid and is sealed to reaction member, is used for PCR Amplification；

The reaction condition of PCR amplification: the first stage, 95 DEG C initial denaturation 10 minutes；Second stage, 94 DEG C be denaturalized 30 seconds, 56 DEG C extend 30 seconds, 40 circulation；Phase III, 98 DEG C are stablized micro- reaction 10 minutes；Final 4 DEG C of terminations reaction；

Pcr amplification product is put into Clarity^TMIn reading apparatus, software is opened, completes setting for detection sample essential information It sets；After the completion of detection, checks the value in the channel FAM and VIC channel fluorescence signal, pass through negative control sample, positive control And no template control sample reference and fluorescence signal distribution, micro- reaction signal threshold value is adjusted；

Sensitivity of the invention and mutation recall rate are detected with dPCR system, when above-mentioned positive control mixes When the value of liquid theoretical mutations rate is respectively 10%, 5%, 1%, 0.1%, actually measured mutation rate is as shown in table 8,

8 sensitivity technique result of table

The sensitivity technique result of kit is consistent with theoretical value, shows that primer and probe have preferable specificity, spirit Sensitivity detection is good；When the positive control mutation rate that saltant type is mixed with wild-type nucleic acid is 0.1%, dPCR system can It is positive for mutation that stable detection goes out corresponding type, actually detected mutation rate difference average out to 0.12%, 0.09%, 0.13%, 0.09%, therefore, the mutation rate that detects of the invention is 0.1%.

Fig. 1 shows the dPCR result of the present embodiment detection mutational site C797S negative control.

Fig. 2 shows the dPCR result of the present embodiment detection mutational site C797S gDNA control.

Fig. 3 shows the dPCR result of the present embodiment detection mutational site C797S positive control.

Embodiment 3: accuracy detection

According to the copy number of measured check sample, accuracy reference material is prepared

By gDNA check sample Caco2 cell strain DNA and positive sample H1650 cell strain DNA, H1975 cell strain DNA, Artificial synthesized large fragment 1,2 mixes respectively by a certain percentage, and mutant proportion 5%, positive sample concentration is about 2000copies/ μL；Specifically,

(1) it prepares the site EGFR gene 19del accuracy reference material: taking the gene of gDNA check sample Caco2 cell strain Group DNA and the H1650 cell strain genomic DNA containing the mutational site 19del, mix according to a certain percentage, and 19del mutation is made The mixed liquor of the total mutant proportion 5% of type nucleic acid；

(2) it prepares the site EGFR gene T790M accuracy reference material: taking the gene of gDNA check sample Caco2 cell strain Group DNA and the H1975 cell strain genomic DNA containing the mutational site T790M, mix according to a certain percentage, and T790M mutation is made The mixed liquor of the total mutant proportion 5% of type nucleic acid；

(3) it prepares the site EGFR gene L858R accuracy reference material: taking the gene of gDNA check sample Caco2 cell strain Group DNA and the H1975 cell strain genomic DNA containing the mutational site L858R, mix according to a certain percentage, and L858R mutation is made The mixed liquor of the total mutant proportion 5% of type nucleic acid；

(4) it prepares the site EGFR gene C797S accuracy reference material: taking the gene of gDNA check sample Caco2 cell strain Group DNA and the artificial synthesized large fragment 1,2 containing the mutational site C797S, mix according to a certain percentage, and C797S saltant type is made The mixed liquor of the total mutant proportion 5% of nucleic acid；

Every kind of accuracy reference material is done 2 repetitions and is tested, totally 8 pipe；Take 4 μ of the site EGFR gene 19del accuracy reference material The site L, EGFR gene T790M accuracy reference material 4 μ L, 4 μ L of the site EGFR gene L858R accuracy reference material, EGFR gene Accuracy reference material 4 μ L in the site C797S is loaded into eight connecting legs of the prepared dPCR reaction system of step 2, so that dPCR The total volume of reaction solution is 22 μ L；Eight connecting leg pipe lids are covered tightly, are mixed well, high speed centrifugation 10 seconds, are prepared for micro- reaction；

PCR reaction condition are as follows: the first stage, 95 DEG C initial denaturation 10 minutes；Second stage, 94 DEG C are denaturalized 30 seconds, and 56 DEG C are prolonged It stretches 30 seconds, 40 circulations；Phase III, 98 DEG C are stablized micro- reaction 10 minutes；Final 4 DEG C of terminations reaction；

Pcr amplification product is put into Clarity^TMIn reading apparatus, software is opened, completes setting for detection sample essential information It sets；After the completion of detection, check the fluorescence signal value in the channel FAM and the channel VIC, by gDNA check sample, positive control, The reference of negative control sample and the distribution of fluorescence signal, class adjust micro- reaction signal threshold value；

It is detected with accuracy of the dPCR system to kit of the present invention, obtains result such as table 9,

Table 9:

As a result, the testing result positive rate of each quality-control product accuracy is 100% according to upper table, meet theoretical qualitative mark Standard shows that the accuracy detection of kit of the present invention meets the requirements.

Embodiment 4: clinical application experiment

2~the 10mL of venous blood for extracting 30 NSCLC patients is placed in noninvasive heparin tube, provides 30 patients of blood sample Tissue specimen detection is carried out, and is clearly the carrying mutational site EGFR gene 19del, the mutational site T790M, L858R mutation The one or more in site and the mutational site C797S, or be free of above 4 kinds of mutation types；It carries out sample labeling and ensures to mark It is errorless to sign information, is stored at room temperature, isolates blood plasma in 12 hours, saved backup for extracting free nucleic acid sample or -80 DEG C；Make With Da'an Gene Company, Zhongshan University's article No. be cat.#DA0670 or the blood free nucleic acid of cat.#DA0680 mentions It takes kit to extract sample, is operated according to kit specification；Mentioned nucleic acid is measured with 2.0 fluorescent quantitation instrument of Qubit Purity and concentration, be placed in -20 DEG C of refrigerators and save；

Each 5 μ L of check sample in the 5 μ L of DNA profiling and kit of each sample is taken, the dPCR prepared to step 2 is loaded In eight connecting legs of reaction system, make the 16 μ L of total volume of every pipe dPCR reaction solution；Eight connecting leg pipe lids are covered tightly, are mixed well, it is high Speed centrifugation 10 seconds, prepares for micro- reaction；

Pcr amplification product is put into Clarity^TMIn reading apparatus, software is opened, completes setting for detection sample essential information It sets；After the completion of detection, check the fluorescence signal value in the channel FAM and the channel VIC, by negative control sample, positive control, The reference of no template control sample and the distribution of fluorescence signal, are adjusted micro- reaction signal threshold value；

Testing result are as follows: in 30 samples, 6 samples are positive in 19del, and 10 samples are positive in T790M, 3 samples In the L858R positive, 3 samples are positive in C797S, remaining sample is feminine gender, the result consistency of examined result and tissue biopsy It is 100%.

Fig. 4 shows the dPCR result of the present embodiment detection mutational site C797S typical positive sample DNA template.

Comparative example 1

The present inventor has screened tens of pairs of digital pcr primers in the course of the research, for each mutational site target sequence, Wherein the overwhelming majority is unable to satisfy the demand of digital pcr.For the typical general primer sequence in the mutational site C797S and detection Effect data is as follows:

Compare primer pair 1:

Upstream primer 1:5 '-ACATCCACCCAGATCACTGG-3 ' (SEQ ID NO.:21)；

Downstream primer 1:5 '-AGCAACATCTCCGAAAGCCA-3 ' (SEQ ID NO.:22).

Compare primer pair 2:

Upstream primer 2:5 '-TCACTGGGCAGCATGTGGCAC-3 (SEQ ID NO.:23)；

Downstream primer 2:5 '-TGAGTTTCTGCTTTGCTGTG-3 ' (SEQ ID NO.:24).

Specific detecting step, testing conditions, the same above embodiments of probe sequence, use the testing result of control primer pair 1 As shown in figure 5, testing result shows that the primer pair specificity is very poor.Using control primer pair 2 testing result as shown in fig. 6, Testing result shows that the primer pair specificity is very poor.Control primer pair 1 and control primer pair 2 are unable to satisfy wanting for clinical detection It asks.

Comparative example 2

In the course of the research, for each mutational site target sequence, digital pcr is carried out with probe sequence by the present inventor Optimize repeatedly, although finding that only a poor nucleotide can significantly affect detection specificity to some probes in research, wherein big absolutely Part is unable to satisfy the demand of digital pcr.For the typical average probe sequence and detection effect data in the mutational site C797S It is as follows:

Control probe group 1:

Mutant probe 1-1:5 '-FAM-CTCTGTCATAGGGAC-MGB-3 ' (SEQ ID NO.:25)；

Mutant probe 1-2:5 '-FAM-CTTCGGCAGCCTCCTG-MGB-3 ' (SEQ ID NO.:26)；

Wild probe 1:5 '-VIC-CTCTGCCATAGGGA-MGB-3 ' (SEQ ID NO.:27).

Control probe group 2:

Mutant probe 2-1:5 '-FAM-ATGCCCTTCGGCTCCCT-MGB-3 ' (SEQ ID NO.:28)；

Mutant probe 2-2:5 '-FAM-TCTCTCTCTGTCATAG-MGB-3 ' (SEQ ID NO.:29)；

Wild probe 2:5 '-VIC-CTCTCTCTGCCATAG-MGB-3 ' (SEQ ID NO.:30).

Specific detecting step, testing conditions, the same above embodiments of primer sequence, using control probe 1 testing result such as Shown in Fig. 7, testing result shows that the specificity of control probe group 1 is poor, sensitivity is poor.Use the detection knot of control probe 2 Fruit as shown in figure 8, testing result show according to probe groups 2 specificity it is poor, sensitivity is poor.Control probe group 1 and shine probe Group 2 is unable to satisfy the requirement of digital pcr detection clinical application.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

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Claims

1. a kind of primer pair for digital pcr detection 20 exon genes of EGFR gene mutation, which is characterized in that the primer To including upstream primer and downstream primer；Wherein, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.:16, institute The nucleotide sequence of downstream primer is stated as shown in SEQ ID NO.:17.

2. a kind of probe groups for digital pcr detection 20 exons mutation of EGFR gene, which is characterized in that the probe groups packet Include nucleotide sequence first mutant probe as shown in SEQ ID NO.:18 and/or nucleotide sequence such as SEQ ID NO.:19 Shown in the second mutant probe；Preferably, the probe groups further include that nucleotide sequence is wild as shown in SEQ ID NO.:20 Probe.

3. the probe groups for digital pcr detection 20 exons mutation of EGFR gene, feature exist as claimed in claim 2 In 5 ' ends of first mutant probe and second mutant probe include fluorescent reporter group FAM；And/or described 3 ' ends of one mutant probe and second mutant probe include fluorescent quenching group MGB；Preferably, the wild probe 5 ' ends include fluorescent reporter group VIC；And/or 3 ' ends of the wild probe include fluorescent quenching group MGB.

4. a kind of kit for digital pcr detection EGFR genetic mutation, which is characterized in that the kit draws including first Object pair, the first primer is to for primer pair described in claim 1.

5. kit as claimed in claim 4, which is characterized in that the kit further includes the first probe groups, and described first Probe groups are probe groups as claimed in claim 2.

6. kit as claimed in claim 4, which is characterized in that the kit further includes the second primer pair, and described second Primer pair includes downstream primer shown in upstream primer shown in SEQ ID NO.:8 and SEQ ID NO.:9；And/or

The kit further includes third primer pair, the third primer pair include upstream primer shown in SEQ ID NO.:1 and Downstream primer shown in SEQ ID NO.:2；And/or

The kit further includes the 4th primer pair, and the 4th primer pair includes upstream primer shown in SEQ ID NO.:12 With downstream primer shown in SEQ ID NO.:13；And/or

The kit further includes the 5th primer pair, the 5th primer pair include upstream primer shown in SEQ ID NO.:5 and Downstream primer shown in SEQ ID NO.:6.

7. kit as claimed in claim 4, which is characterized in that the kit further includes the second probe groups, and described second Probe groups include mutant probe shown in SEQ ID NO.:10；And/or wild probe shown in SEQ ID NO.:11；And/or

The kit further includes third probe groups, and the third probe groups include the resistance of PCR amplification shown in SEQ ID NO.:3 Disconnected agent, wild probe shown in mutant probe and/or SEQ ID NO.:7 shown in SEQ ID NO.:4；And/or

The kit further includes the 4th probe groups, and the 4th probe groups include mutant probe shown in SEQ ID NO.:14； And/or wild probe shown in SEQ ID NO.:15.

8. a kind of method of digital pcr detection EGFR genetic mutation, which is characterized in that the method includes the steps:

(1) DNA sample of object to be detected is provided；

Wherein, the digital pcr reaction system includes the first digital pcr reaction system, the first digital pcr reaction system packet It includes described in the DNA sample of step (1) offer, primer pair and second aspect of the present invention described in first aspect present invention Probe groups.

9. method according to claim 8, which is characterized in that the digital pcr reaction system further includes that the second digital pcr is anti- Answer system, the second digital pcr reaction system include step (1) provide the DNA sample, second primer pair and Second probe groups；And/or

The digital pcr reaction system further includes third digital pcr reaction system, and the third digital pcr reaction system includes The DNA sample, the third primer pair, five primer pair and the third probe groups that step (1) provides；And/or

The digital pcr reaction system further includes the 4th digital pcr reaction system, and the 4th digital pcr reaction system includes The DNA sample, the 4th primer pair and the 4th probe groups that step (1) provides.

10. the purposes of primer pair described in claim 1, and/or probe groups as claimed in claim 2, which is characterized in that be used for Digital pcr detection kit is prepared, the digital pcr detection kit is for detecting 20 exons mutation of EGFR gene.