CN107557469A - A kind of specific primer in detection KRAS gene G12C sites is to, probe and kit - Google Patents

Abstract

The invention provides a kind of specific primer in detection KRAS gene G12C sites to, probe and kit, the nucleotide sequence of specific primer of the invention is as shown in SEQ ID NO.1 2, and the nucleotide sequence of probe is as shown in SEQ ID NO.3 4.Primer of the present invention is designed based on KRAS gene G12C sites, utilize above-mentioned specific primer pair and probe, KRAS gene G12C sites in poba gene group DNA or dissociative DNA to be measured are detected using the method for digital pcr, specific good and high sensitivity, testing result accuracy is high, the frequency of mutation based on site can be directly given simultaneously in detection mutation, there is important application value.

Description

A kind of specific primer in detection KRAS gene G12C sites is to, probe and kit

Technical field

The present invention relates to technical field of gene detection, specifically, is related to a kind of detection proto-oncogene KRAS gene G12C The specific primer in site is to, probe and the kit containing it.

Background technology

KRAS gene codes relate generally to adjust fissional K-Ras protein.As by RAS/MAPK approach A part for signal pathway, protein is by the nucleus from extracellular signal transduction to cell.These signal designation cells Growth and division or maturation simultaneously play special function (differentiation).K-Ras albumen is a kind of GTP enzymes, it means that it is by GTP's Molecule is converted into GDP another molecule.As a switch, it is opened and closed K-Ras protein by GTP and GDP molecules.For Transmission signal, K-Ras albumen must be opened to GTP molecules by adhering to (with reference to).When GTP is converted into by K-Ras albumen During GDP, it is closed (inactivation).When protein is combined with GDP, it will not transmit signals to the core of cell.KRAS genes Belong to the gene of a kind of oncogene.When mutation, oncogene has the potentiality for causing normal cell to be changed into cancer.KRAS genes exist In the Ras families of oncogene, it also includes two other genes：HRAS and NRAS.These protein are in cell division, cell point Change and cell self is destroyed in (Apoptosis) and played an important role.

Proto-oncogene KRAS is one of important molecule in EGFR signal paths, is represented according to conventional research, saltant type KRAS genes are active all the time, are not influenceed by upstream EGFR gene state, only wild type (unmutated type) KRAS Gene is influenceed by upstream EGFR signal stimulus, and this is also that EGFR inhibitor (TKIs) is controlled with saltant type KRAS genes patient Treat invalid theoretical foundation.KRAS gene mutation can cause cell evasion apoptosis, and this exception is in cancer of pancreas, colorectal cancer, lung Incidence in the malignant tumours such as cancer is higher.In cancer of pancreas, the point mutation incidence of KRAS genes is up to more than 90%.

US National synthesis cancer network (NCCN)《Colorectal cancer clinical practice guideline》(V3.2011) explicitly pointed out in： (1) all metastatic colorectal cancer patients should all detect KRAS gene appearances；(2) KRAS wild types patient just suggests receiving EGFR inhibitor (such as Erbitux and Victibix) is treated.NCCN simultaneously《Non-small cell lung cancer clinical practice guideline》 (V2.2011) also indicated that in：When KRAS genes are undergone mutation, it is not recommended that use EGFR-TKIs target therapeutic agents.In addition, What the Ministry of Public Health of China promulgated《Colorectal cancer diagnosis and treatment specification》In clearly propose：(1) colorectal cancer row histopathologic examination, it is determined that For recurrence or metastatic colorectal carcinoma when, detect tumor tissues K-ras gene appearances.(2) pathology of surgery for colorectal carcinoma sample It should include K-ras gene appearances in report content and requirement, be defined as recurrence or metastatic colorectal carcinoma.Such as without surgery excision mark Originally can be determined from biopsy specimen.(3) tumour K-ras gene appearances are detected before late period/metastatic colorectal carcinoma chemotherapeutic treatment, EGFR is not recommended for as routine inspection project.U.S. FDA carries out the genetic tests such as EGFR, KRAS before having Qiang Zhiyaoqiud medication. At home, the development of KRAS genetic tests obtains advocating for Chinese Anti-Cancer Association.In this context, it is domestic to have carried out KRAS in succession The detection of gene and corresponding treatment work, KRAS genetic tests have gradually developed into one of malignant tumour conventional detection project.

At present, the detection method about KRAS is a lot, and most gene mutation detection techniques are all based on PCR progress 's.Grown using the method cycle of two generations sequencing, expense is of a relatively high；It is glimmering although shorter using the cycle of quantitative fluorescent PCR The technology of Fluorescent Quantitative PCR belongs to relative quantification, it is necessary to carry out the calculating of specific concentration by standard curve to a certain extent, essence Exactness is limited；The Observation On The Prognosis cycle using digital pcr progress anaphase is short, and cost is low and targeted.

Digital pcr (Digital-PCR) is a kind of new PCR detection method, and its operation principle is DNA or cDNA Sample segment is many independent, parallel PCR reaction systems, and partly these, which react, contains target molecules (positive), and other Not comprising (feminine gender).Individual molecule can be amplified 1,000,000 times or more, it is possible to increase the performance of existing TaqMan measure, from And higher sensitivity, the degree of accuracy are realized, avoid the appearance of false negative result.Standard PCR reaction systems after droplet occurs, It is assigned in about 20000 droplets, each droplet includes or the target molecule (DNA profiling) not comprising one or more copy, Realize " unimolecule template PCR amplifications ", after amplification terminates, the droplet containing nucleic acid templates can provide fluorescence signal, not have The droplet of template just produces without fluorescence signal.Finally according to Poisson distribution principle and the ratio of positive droplet, analysis software The concentration or copy number for providing target molecule to be checked can be calculated.Digital pcr can directly calculate the copy number of target sequence, therefore nothing Control sample and standard curve need to be depended on to can be carried out accurate absolute quantitation detection；Further, since digital pcr is being carried out As a result only judge during interpretation with/without two kinds of amplification states, therefore also without the intersection point of detection fluorescence signal and given threshold line, The identification of Ct values is completely independent of, therefore the reaction of digital pcr is influenceed to substantially reduce by amplification efficiency, and PCR is reacted and pressed down The tolerance of thing processed greatly improves；The process of digital pcr experiment Plays reaction system distribution can be reduced largely With the background sequence concentration of the competitive effect of target sequence, the concentration or copy number for providing target molecule to be checked are calculated.

The content of the invention

It is an object of the invention to provide a kind of side in low, degree of accuracy high sensitivity the detection KRAS gene G12C sites of cost Method.

Present invention firstly provides a pair of specific primers pair for being used to detect KRAS gene G12C sites, the specificity is drawn The nucleotide sequence of thing pair is as shown in SEQ ID NO.1-2.

Forward direction primer：5’-GCCTGCTGAAAATGACTGAATATAAACT-3′(SEQ ID NO.1)

Reverse primer:5’-GCTGTATCGTCAAGGCACTCTT-3′(SEQ ID NO.2)

The invention provides with above-mentioned specific primer to the use of probe combinations, the nucleotides of the probe combinations Sequence is as shown in SEQ ID NO.3-4.

Further, 5 ' mark VIC, 3 ' mark MGB-NFQ, shown in SEQ ID NO.4 of probe shown in SEQ ID NO.3 5 ' flag F AM, 3 ' mark MGB-NFQ of probe.

The probe combinations that the present invention designs, particular sequence are

VIC：5’-VIC-TTGGAGCTGGTGGCGTA-MGB-NFQ-3′；

FAM：5′-FAM-TTGGAGCTTGTGGCGTA-MGB-NFQ-3′.

Containing the kit of probe shown in specific primer pair shown in SEQ ID NO.1-2 and/or SEQ ID NO.3-4 or Detection reagent belongs to the protection domain of the application.

The invention provides above-mentioned specific primer pair and probe combinations or containing its kit in detection KRAS genes Application in G12C sites.

The kit of the present invention, its working procedure are：

(1) genomic DNA or dissociative DNA of blood sample to be measured are extracted；

(2) using the genomic DNA of extraction or dissociative DNA as template, using the specific primer pair described in claim 1 and Probe combinations described in claim 2, carry out digital pcr amplified reaction；

(3) the copy number result of determination provided according to fluorescence signal.

The program of wherein step (2) digital pcr amplified reaction is：95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 20s, 58 DEG C are moved back Fire and extension 45s, totally 40 circulations；98 DEG C of solidification droplet 10min.

The present invention provides a kind of method for detecting KRAS gene G12C sites in genomic DNA or dissociative DNA in blood, bag Include following steps：

(1) genomic DNA or dissociative DNA of blood sample to be measured are extracted；

(2) using the genomic DNA of extraction or dissociative DNA as template, using the specific primer pair described in claim 1 and Probe combinations described in claim 2, carry out digital pcr amplified reaction；

(3) the copy number result of determination provided according to fluorescence signal.

In the above method, the program that digital pcr amplified reaction is carried out in step (2) is：95 DEG C of pre-degeneration 10min；94℃ It is denatured 20s, 58 DEG C of annealing and extension 45s, totally 40 circulations；98 DEG C of solidification droplet 10min.

The invention provides for detect in poba gene group DNA or dissociative DNA the method in KRAS genes G12C sites and Detection kit, the selection of cancer patient's therapeutic scheme suitable for detecting EGFR mutation.The present invention uses droplet digital pcr Technology, a pair of specific primers pair and two probes being used cooperatively with it are designed for KRAS gene G12C site mutations.By It is one of important molecule in EGFR signal paths in proto-oncogene KRAS, is represented according to conventional research, saltant type KRAS bases Because being active all the time, do not influenceed by upstream EGFR gene state, only wild type (unmutated type) KRAS genes by The influence of upstream EGFR signal stimulus, this is also invalid to EGFR inhibitor (TKIs) treatment with saltant type KRAS genes patient Theoretical foundation.Patient can be guided target medication and help prevent patient carrying out invalid drug therapy.

It is applicable not only to provided by the present invention for the method for detecting KRAS genes G12C to tumor tissues, FFPE group The nucleic acid for knitting source is detected, and is more applicable for the inspection to the sites being mutated of KRAS genes G12C in blood circulation free nucleic acid Survey；And the combination detection architecture and droplet type digital pcr system can accurately be detected to the frequency of mutation of sample of nucleic acid.This hair The specific primer and probe of bright offer, poba gene group DNA or dissociative DNA can accurately be detected, are mutated in detection The frequency of mutation of gene loci can be directly given simultaneously, and combine digital PCR system to the detection of mutation up to 5,000/ One, high sensitivity.

Brief description of the drawings

Fig. 1 is finally to determine annealing temperature for the selection result figure of annealing temperature in digital pcr course of reaction of the present invention 60 DEG C are optimum annealing temperature.

When Fig. 2 is that mutant plasmids loading concentrations are from 0.00001 to 0.0000001ng/ul in the embodiment of the present invention 3 (A03,04；B03,04；C03,04), mutant probe in detecting copies from 18.1-979.

When Fig. 3 is that wild plasmid loading concentrations are from 0.00001 to 0.0000001ng/ul in the embodiment of the present invention 3 (D03,04；E03,04；G03, F04), the average copy numbers of WT probe in detecting two multiple holes in 20ul is from 17.2 to 1255 Copy.

Fig. 4 (saltant type) is mixed type plasmid (saltant type in the embodiment of the present invention 3:Wild type=1：1) loading concentrations from 0.00001 (F03, G04 when arriving 0.0000001ng/ul；H03,04；A05,06), saltant type probe two multiple holes in 20ul Average copy number detection is from 18-902.

Fig. 5 (wild type) is mixed type plasmid (saltant type in the embodiment of the present invention 3:Wild type=1：1) loading concentrations from 0.00001 (F03, G04 when arriving 0.0000001ng/ul；H03,04；A05,06), wild-type probe detection is answered for two in 20ul The average copy number in hole copy from 14.3 to 826.

Fig. 6 is the wild of seven tumour patient dissociative DNAs of the degree of accuracy and clinical practice test in the embodiment of the present invention 3 Type testing result (A08, B08, C08, D08, E08, F08, G08), detection find that 7 people have wild type copies detection, copy number .

Fig. 7 is the mutation of seven tumour patient dissociative DNAs of the degree of accuracy and clinical practice test in the embodiment of the present invention 3 Type testing result (A08, B08, C08, D08, E08, F08, G08), detection find only have sample one (A08) to have saltant type copy inspection Go out.

Embodiment

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.

Experimental method in following embodiments, it is conventional method unless otherwise specified.

Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.

Embodiment 1 is used for the design of the specific primer pair for detecting KRAS gene G12C sites and specific probe combination

1st, design of primers：The DNA sequence dna of people's KRAS genes is searched from NCBI, is gone out using primer6.0 Software for Design It can amplify comprising mutational site (c.34G>T) the upstream and downstream primer of fragment, 4 are therefrom chosen to (being respectively SEQ ID NO.1- 2, SEQ ID NO.1 and 5, SEQ ID NO.1 and 6, SEQ ID NO.1 and 7), the clip sizes of 4 pairs of primers amplifications is from 200bp To 50bp；Synthesize 4 pairs of primers；, 4 pairs of primers are tested using round pcr.

QPCR experiments are carried out to gained primer, verify the amplification efficiency of different primers group, QPCR results show identical amplification Under system and amplification condition, QPCR CT values occur earliest when primer sets amplified production length is 67bp, show the effect of amplification Rate is optimal, therefore have selected pair of primers group (reverse primer 1 and forward direction primer) of the amplification length in 67bp, its nucleotide sequence For：

Forward direction primer：5’-GCCTGCTGAAAATGACTGAATATAAACT-3′

Reverse primer 1:5’-GCTGTATCGTCAAGGCACTCTT-3′

Reverse primer 2：5’-CTGTATCAAAGAATGGTCCTGC-3′

Reverse primer 3：5’-GCTGTATCGTCAAGGCACT-3′

Reverse primer 4：5’-TGTATCGTCAAGGCACTCTT-3′

And concentration optimization is carried out to the primer that screening obtains, forward direction primer and reverse primer set different concentration respectively, If final concentration is from 50nM to 500nM, is verified using QPCR, as a result show primer working concentration in 200nM, QPCR CT Value occurs earliest, therefore screening obtains the final concentration of 200nM of most suitable primer.

2nd, probe designs：For screening obtained specific primer pair, design to obtain through primer design and coordinate with it The specific probe used, it can be used together when detecting KRAS gene G12C mutational sites.

Because MGB groups have high compatibility to DNA double chain ditch, therefore in PCR reactions, MGB probes compared to Average probe Tm values are much higher, to be generally higher by 10-20 DEG C.MGB probes have high Tm values, can be very good to overcome GC to contain Difficulty of the low template when probe designs is measured, waits the Tm values between site mutation sequence widely different, improves the special of detection Property.Meanwhile MGB probes include a not fluorescent quenching group (NFQ), it can be really eliminated caused by traditional quenching group Background fluorescence, signal to noise ratio is improved, so as to lift detection sensitivity.

The nucleotides sequence of the probe is classified as：

VIC：5’-VIC-TTGGAGCTGGTGGCGTA-MGB-NFQ-3′；(SEQ ID NO.3)

FAM：5′-FAM-TTGGAGCTTGTGGCGTA-MGB-NFQ-3′(SEQ ID NO.4).

And concentration optimization is carried out to the probe of design：Use the primer of above-mentioned screening and the concentration of optimization, template concentrations point Wei not 0.1ng/ul, 0.01ng/ul, 0.001ng, 0.0001ng/ul and 0.00001ng/ul.Using different concentration and probe concentrations, Such as it is respectively that 100nM, 200nM and 300nM are detected respectively to the template of 3 concentration using QPCR, probe is worked as in as a result display When concentration is 200nM, the CT values that the QPCR of low concentration template occurs are earliest, therefore the final concentration of 200nM of most suitable probe.

Embodiment 2 detects the foundation of the digital pcr detection method in KRAS gene G12C sites

1st, prepare reaction system to be determined in optimal primer and probe according to embodiment 1, determine reaction system 20ul, 2 × dd To primer 1.8ul before PCR premixed liquids 10ul, 10uM, 10uM reverse primers 1.8ul's, 10uM is wild with VIC fluorescence labelings Type probe (SEQ ID NO.3) 0.5ul, 2uM saltant type probe (SEQ ID NO.4) 0.5ul with FAM marks, water 3.4ul, template 2ul, common 20ul.

2nd, after completing step 1, reaction system is transferred in the system well of digital pcr droplet generation card, and anti- Using the digital pcr reaction oil that 70ul is added in oily well, digital pcr droplet generation card is transferred to Droplet In Generator drop generators, start instrument, obtain micro system.

PCR reaction solutions are prepared, are stored at room temperature after configuration 3 minutes.PCR reaction solutions after standing are transferred to DG8 droplets hair Sample cell in raw card, add droplet and special oil occurs.It is put into QX200Droplet Generator and carries out droplet Reaction.Generated droplet is shifted to 96 orifice plates.It is covered with PCR viscosity envelope paillon foil and carries out sealer using heat-sealing instrument.It is subsequently transferred to PCR to carry out amplified reaction, reaction condition be 95 DEG C 10 minutes；94 DEG C 20 seconds, (B03：56.6 C03：58.2 D03：60.1, E03：61.7 F03：62.6) DEG C 45s, 40 circulations；98 DEG C 10 minutes；12 DEG C continue.It is put into after the completion of reaction QX200Droplet Reader carry out signal-obtaining, according to the result figure of QuantaSoft softwares select optimum annealing temperature for 60 DEG C, see Fig. 1.Data are analyzed according to the result figure of QuantaSoft softwares, wherein the frequency of mutation=saltant type copy Number/total copy number (saltant type copy number+wild type copies number)

The method that embodiment 3 is established according to embodiment 2 carries out specificity and sensitivity technique

1st, specificity and sensitivity experiment

The mutant plasmid (mutant) and include the site wild type that structure includes KRAS gene G12C mutational sites respectively Plasmid (WT), and using the two plasmids as template, the specificity of probe and sensitivity are carried out with droplet type digital pcr system Test.Reaction system includes 2 × mix buffer 10ul, template 2ul (wild plasmid WT 0.00001ng/ul；WT 0.000001ng/ul,WT 0.0000001ng/ul；Mutant plasmids M 0.00001ng/ul；M 0.000001ng/ul, M 0.0000001ng/ul), forward direction primer 1.8ul, reverse primer 1.8ul, WT probe 0.5ul, mutant probe 0.5ul, it is ultrapure Water 3.4ul, common 20ul reaction systems.Reaction condition is 95 DEG C, 10 minutes；94 DEG C, 20 seconds, 58 DEG C, 45s (40 circulations)；98 DEG C, 10 minutes；4℃.

Testing result is as shown in Figure 2 to 3.During wild plasmid (WT) final concentration of 0.00001ng, WT probe in detecting arrives 979 copies, mutant probe is without detection (Fig. 2)；During the final concentration of 0.00001ng of mutant plasmids, wild-type probe without detection, Mutant probe detects 1255 copies；During the final concentration of 0.000001ng of mutant plasmids, WT probes are without detection, mutant probe inspection Go out 143 copies (table 1).Table 1 is to include the mutant plasmid (mutant) in KRAS gene G12C mutational sites in the present embodiment It is template with the plasmid (WT) comprising the site wild type, with specificity of the droplet type digital pcr system to probe and sensitivity Carry out test result.

Table 1

In summary, wild-type probe will not identify G12C mutational site, and saltant type probe will not also identify non-dash forward Become site；And wild type and saltant type probe be on the premise of plasmid final concentration is into 10 times of reductions, its copy number detected also into 10 times of reduction, it was demonstrated that the probe has good specificity.

2nd, mutation rate detection experiment

Mutant plasmids and wild plasmid are pressed 1:1 ratio mixing, loading concentrations are respectively 10^-5ng/ul：10^{- 5}Ng/ul, 10^-6ng/ul：10^-6Ng/ul, 10^-7ng/ul：10^-7ng/ul；With droplet type digital pcr system to the inventive method Mutation detector efficiency is detected, and the melting concn for as a result showing saltant type and wild plasmid is 10^-7When digital pcr detection it is prominent The ratio of modification and wild type is 1:1, and the number of copy number also with concentration in gradient relation, therefore the present invention method pair The detection of mutation can reach the copy number (Fig. 4-Fig. 5, table 1) of units.

Separately by mutant plasmids and wild plasmid according to 1:1,1:10,1:100,1:5000,1:10000 ratio is mixed Close, detected with mutation detector efficiency of the droplet type digital pcr system to the inventive method, as a result show droplet type numeral PCR detector efficiencies are probably at five one thousandths (table 2)

Table 2

3rd, the degree of accuracy and clinical practice test

Intravenous blood collection is carried out to 7 tumor patients, and blood plasma is separated.Circulation trip is carried out with the blood plasma after separation The extraction of freestone acid, is examined with the primer and probe of the invention to KRAS G12C sites respectively with circulating the sample of dissociative DNA Survey, while the method being sequenced using two generations is detected to KRAS G12C sites, testing result is as shown in the figure.Use the present invention Method testing result to there are wild type copies to detect (Fig. 6) in 7 detection samples, but only sample one has KRAS G12C There is (Fig. 7) in site mutation, the frequency of mutation 24.4%；The method being sequenced with two generations detects to same 7 samples, as a result Show that only 1 sample has G12C mutation and existed, the frequency of mutation is 17.15% (table 3).In summary, method energy of the invention Enough accurate detection samples can calculate the frequency of mutation with the presence or absence of the presence of KRAS gene G12C site mutations, and with two generations The frequency of mutation of sequence measurement detection has relative uniformity.

Table 3

The above results show, specific primer (the SEQ ID in the detection KRAS gene G12C sites that embodiment 1 is provided NO.1-2) and probe (SEQ ID NO.3-4) Detection results it is best, suitable for clinical sample to tissue or peripheral blood The detection in KRAS gene G12C sites.

Sequence table

<110>Guangzhou Man Rui biology information technologies Co., Ltd

<120>A kind of specific primer in detection KRAS gene G12C sites is to, probe and kit

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

gcctgctgaa aatgactgaa tataaact 28

<210> 2

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

gctgtatcgt caaggcactc tt 22

<210> 3

<211> 17

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

ttggagctgg tggcgta 17

<210> 4

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<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

ttggagcttg tggcgta 17

<210> 5

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

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ctgtatcaaa gaatggtcct gc 22

<210> 6

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

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gctgtatcgt caaggcact 19

<210> 7

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

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tgtatcgtca aggcactctt 20

Claims (10)

1. a kind of specific primer pair for being used to detect KRAS gene G12C sites, the nucleotide sequence of the specific primer pair As shown in SEQ ID NO.1-2.

2. with claim 1 described in specific primer to the use of probe combinations, the nucleotides sequence of the probe combinations Row are as shown in SEQ ID NO.3-4.

3. probe combinations as claimed in claim 2,5 ' mark VIC, 3 ' mark MGB-NFQ of probe shown in SEQ ID NO.3, 5 ' flag F AM, 3 ' mark MGB-NFQ of probe shown in SEQ ID NO.4.

4. the kit containing probe combinations described in specific primer pair described in claim 1 and/or claim 2.

5. the detection reagent containing probe combinations described in specific primer pair described in claim 1 and/or claim 2.

6. any described probe combinations of specific primer pair and claim 2-3 or claim 4 institute described in claim 1 Application of the detection reagent described in kit or claim 5 stated in detection KRAS gene G12C sites.

7. kit as claimed in claim 4, it is characterised in that its working procedure is：

(1) genomic DNA or dissociative DNA of blood sample to be measured are extracted；

(2) using the genomic DNA of extraction or dissociative DNA as template, the specific primer pair and right described in claim 1 are utilized It is required that the probe combinations described in 2, carry out digital pcr amplified reaction；

(3) the copy number result of determination provided according to fluorescence signal.

8. kit as claimed in claim 7, it is characterised in that the digital pcr amplified reaction of step (2) in its working procedure Program be：95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 20s, 58 DEG C of annealing and extension 45s, totally 40 circulations；98 DEG C of solidifications are micro- Drip 10min.

9. a kind of method in KRAS gene G12C sites in detection poba gene group DNA or dissociative DNA, it is characterised in that including Following steps：

(1) genomic DNA or dissociative DNA of blood sample to be measured are extracted；

(2) using the genomic DNA of extraction or dissociative DNA as template, the specific primer pair and right described in claim 1 are utilized It is required that the probe combinations described in 2, carry out digital pcr amplified reaction；

(3) the copy number result of determination provided according to fluorescence signal.

10. according to the method for claim 9, it is characterised in that the program of digital pcr amplified reaction is carried out in step (2) For：95 DEG C of pre-degeneration 10min；94 DEG C of denaturation 20s, 58 DEG C of annealing and extension 45s, totally 40 circulations；98 DEG C of solidification droplets 10min。