CN102808027B

CN102808027B - EGFR (epidermal growth factor receptor) gene mutation site detection kit

Info

Publication number: CN102808027B
Application number: CN201210291816.5A
Authority: CN
Inventors: 王弢; 秦勇
Original assignee: SUZHOU MICRO DIAG BIOMEDICINE CO Ltd
Current assignee: Jiangsu is the real biopharmaceutical technology Limited by Share Ltd
Priority date: 2012-08-16
Filing date: 2012-08-16
Publication date: 2015-05-13
Anticipated expiration: 2032-08-16
Also published as: CN102808027A

Abstract

The invention discloses an EGFR (epidermal growth factor receptor) gene mutation site detection kit which detects EGFR gene mutation sites by combining the ARMS (amplification refractory mutation system) specific mutant enrichment technology with the Taqman-MGB (minor groove binder) probe specific detection technology. The kit comprises ARMS primer pairs and corresponding Taqman-MGB probes, and the ARMS primer pairs and the corresponding Taqman-MGB probes are used for detecting the EGFR gene mutation sites. The EGFR gene mutation site detection kit is high in sensitivity, capable of detecting multiple samples simultaneously and low in cost, provides guidance for pharmacy of clinicians, realizes individualized treatment of patients suffering from tumors and is capable of reducing treatment risk and burden of the patients and wide in application prospect.

Description

Detect the test kit in EGFR genetic mutation site

Technical field

The present invention relates to biotechnology and medical field, be specifically related to a kind of test kit detecting EGFR genetic mutation site.

Background technology

EGFR is the expression product of proto-oncogene c-erbB1, and be one of 4 members in Epidermal Growth Factor Receptor Family (HER), HER family plays important regulating effect in cellular physiological processes.The transmembrane glycoprotein of EGFR to be molecular weight be 170kD, is made up of extracellular region, cross-film district and intracellular region 3 part; Nitrogen end extracellular region can be divided into again 4 subprovinces, is the position that EGFR and part (transforminggrowthfactor-α, Urogastron, two regulin etc.) combine; Cross-film district is made up of a lipotropy polypeptide spiral; Intracellular region contains conservative tyrosine kinases phosphorylate site.EGFR is monomer under inactive state, and when forming homology or heterodimer after binding of receptor and ligand, acceptor intracellular region generation self-phosphorylation also opens a series of intracellular signal cascade of beginning.Associated signal pathway and signal transmission albumen have: Phospholipase C-γ 1, phosphinositides 3 kinases, serine threonine protein kinase etc.Final activation related nucleoprotein, promotes that cell is transitioned into the S phase from the G phase.Correlative study proves, EGFR plays an important role to cell-derived, EGFR in development of cancer as played an important role in the processes such as vasculogenesis and Nasopharyngeal neoplasms, adhesion and Apoptosis inhibitor.Transforminggrowthfactor-α and EGFR process LAN in solid tumor, often indicates that the state of an illness further develops and poor prognosis (Yarden Y.Nat Rev Mol Ce U Biol, 2001,2 (2) 127-137).

EGFR is distributed widely in the cell surfaces such as mammalian epithelial cell, inoblast, spongiocyte, keratinocyte, and EGFR signal path plays an important role to the growth of cell, the physiological process such as propagation and differentiation.In the protein tyrosine kinase afunction such as EGFR or its associated signal paths the activity of key factor or cellular localization abnormal, all can cause the generation of tumour, diabetes, immune deficiency and cardiovascular disorder.

EGFR is present in most cells, expression is had in kinds of tumors, as colorectal cancer, mammary cancer, carcinoma of the pancreas, prostate cancer and nonsmall-cell lung cancer etc., be wherein 25% ~ 77%(MendelsohnJ in the positive expression rate of colorectal cancer, .J Clin Oncol, 2003, 21 (14): 2787-99), in the propagation that the positive expression rate of cancer of the stomach is this unconventionality expression of 38% ~ 51.5%. and tumour cell, new vessel is formed, invasion and attack, transfer and anti-apoptotic etc. are relevant, the EGFR of overexpression usually imply that disease free survival phase and Overall survival decline, the risk of recurrence and distant metastasis increases (Opdwyer PJ, SeminOncol, 2002, 29).Express the cancer of the stomach poor prognosis of EGFR and part TGF-α thereof simultaneously; within 5 years, survival rate only has 12%; and EGFR and TGF-alpha expression level is normal or only have the cancer of the stomach of a certain overexpression; within 5 years, survival rate then has 45% and 36%(Shah MA; .Proc Am Soc Clin Oncol; 2005,24:abstr4025).Radinsky etc. have detected the EGFR expression of sample after colorectal neoplasm operation, find that the level of the EGFR mRNA of advanced CRC (Dukes ' D phase, hepatic metastases) is 10 ~ 20 times of early lesion.Nonsmall-cell lung cancer (non-smallcell lung cancer.NSCLC) up-regulated expression EGF-R ELISA (the epidermal growth factor receptor of about 40% ~ 80%, EGFR), EGFR signalling system participates in tumor invasion, vasculogenesis, apoptosis obstacle and chemoradiotherapy and resists, and is that NSCLC treats one of important target spot.Gefitinib is the inhibitor of EGFR Tyrosylprotein kinase (TK), and II clinical trial phase shows, has good curative effect to part NSCLC, and the remission rate of second line treatment is 10% ~ 19%.Research shows, the curative effect of Gefitinib is relevant with EGFR acceptor gene the 18th, 19,20 and 21 exons mutation, to the many sudden changes with EGFR gene of Gefitinib reaction patient.Wherein the sudden change of more than 90% is present in the 19th exon and the 21st exon.Therefore, set up the method for rapid detection NSCLC tumor tissue EGFR genetic mutation, contribute to screening patient and treat.

The epidermal growth factor recipient tyrosine kinase inhibitor (EGFR-TKI) being representative with Iressa (Iressa), occur by Tumor suppression, necessary epidermal growth factor recipient tyrosine kinase blocks tumour cell in development intracellular signaling, thus reach the hyperplasia of inhibition tumor cell, invasion and attack, transfer, vasculogenesis promote that the tune of tumour cell is died.Iressa is a kind of oral molecular targeted therapy, it is EGF-R ELISA (EGFR) tyrosinase inhibitor (TKI), because EGFR-TK and signal path thereof are at nonsmall-cell lung cancer (non-small-cell lung cancer, NSCLC) play an important role in generation, development, so to treat tumour be a focus by blocking it.Iressa in Discussion on Chinese Listed, was the surface growth factor receptors-tyrosine kinase inhibitor uniquely having the clinical data contrasted with standard second-line chemotherapy at present in 2005.There is very large individual difference to the curative effect of nonsmall-cell lung cancer in clinical practice display Iressa, only has the individuality of 8-18% to have very good effect.

The EGFR genetic mutation detection method generally applied at present is restriction small segment length polymorphism analysis method (RFLP method) and DNA direct Sequencing etc.RFLP method is the method combined with restriction enzyme digestion by PCR.But the method exists following shortcoming: RFLP experimental implementation is loaded down with trivial details, and sense cycle is long, with high costs, there is first round enzyme and cut the false positive not exclusively caused, non-stopped pipe operation, is easy to pollute, is difficult to meet clinical detection requirement.The principle of DNA direct Sequencing is: it is longer that the method exists following shortcoming sense cycle, both expensive, and non-stopped pipe operation, be difficult to avoid crossed contamination, flux is not high.The sensitivity of DNA direct Sequencing is lower, the problems such as the existence of the contracting of heterozygous mutant, glue laminated, GC enrichment region make to be difficult to obtain accurate data by once sequencing, need repeatedly to repeat order-checking and just may avoid false positive etc., therefore direct Sequencing method difficulty is applicable to clinical detection.

And clinical a kind of quick, accurate, easy and simple to handle and avoid the test kit of detection EGFR genetic mutation of crossed contamination in the urgent need to developing, meet the ageing requirement of clinical application and checkout and diagnosis aspect.

Taqman probe is the detection technique of fluorescence gone out at Real-time round pcr platform development, and 5 ' end of probe is containing fluorescent reporter group, and 3 ' end is containing fluorescent quenching group.When probe is complete, the fluorescent signal that reporter group is launched is quenched group absorptions, when carrying out pcr amplification, probe enzyme is cut degraded to the 3 ' 5 prime excision enzyme activity held by 5 ' end of Taq archaeal dna polymerase, reporter group is separated with quenching group, send fluorescent signal, thus reach the accumulation of fluorescent signal and PCR primer forms Complete Synchronization.Taqman-MGB probe is the Taqman probe at 3 ' end with minor groove binding molecule (minor groove binder, MGB), improves the Tm value of probe, shortens probe length, detect while being convenient to multi-mutant site.

Amplification refractory mutation system (amplification refractory mutation system, ARMS) be PCR-based technical foundation grows up for check point sudden change or the technology of polymorphism.ARMS has been successfully applied to analyzing gene polymorphism, comprises germinal mutation and somatic mutation, and this technology can tell the sudden change of low levels under a large amount of wild DNA background.Lack 3 ' end to 5 ' end 5 prime excision enzyme activity according to the Taq archaeal dna polymerase used during pcr amplification, primer 3 ' can not be revised and hold base mispairing, as long as primer 3 ' is held with template 1 base unpaired, just cannot amplify specific product.For this characteristics design primer, saltant type is increased, wild-type cannot increase, thus is exaggerated mutant signal, is convenient to detect.

This technology of Taqman-ARMS, based on Real-time PCR platform, detects two kinds of technology in conjunction with ARMS sudden change enrichment and Taqman-MGB specificity fluorescent.Utilize ARMS primer pair mutated target sequence to carry out pcr amplification, Taqman-MGB probe carries out specific position detection to amplified production, and specific sudden change is identified on Real-time PCR basis.

Summary of the invention

In view of this, an object of the present invention is the test kit providing quick, accurate, easy and simple to handle and antipollution detection EGFR genetic mutation site, solve in prior art carry out that EGFR genetic mutation trace routine is loaded down with trivial details, somewhat expensive, time-consuming, easy pollution and the problem such as sensitivity is low, especially provide atraumatic outside pathological tissue as the detection such as serum or blood plasma.

For achieving the above object, technical scheme of the present invention is:

Detect the test kit in EGFR genetic mutation site, described test kit comprise detect EGFR genetic mutation site ARMS primer pair and specific recognition described in the Taqman-MGB probe in mutational site; Upstream primer or the downstream primer of described ARMS primer pair comprise EGFR genetic mutation site;

The Taqman probe in the amplified production positive-sense strand mutational site of described Taqman-MGB probe specific recognition ARMS primer, 3 ' end of described probe is combined with minor groove binding molecule.

Described mutational site be EGFR gene 2235-2249 position disappearance, 2236-2250 position disappearance, 2236-2253 position disappearance, 2239-2253 position disappearance, 2240-2257 position disappearance, 2582 sudden change and 2573 sudden change in one or more.

Preferably, in described test kit, the primer pair detecting EGFR gene 2235-2249 position disappearance is made up of the downstream primer shown in the upstream primer shown in SEQ ID No:6 and SEQ ID No:7, and the nucleotide sequence of Taqman-MGB probe is as shown in SEQ ID No:21;

The primer pair detecting EGFR gene 2236-2250 position disappearance is made up of the upstream primer shown in SEQ ID No:8 and the downstream primer shown in SEQ IDNo:9, and the nucleotide sequence of Taqman-MGB probe is as shown in SEQ ID No:23;

The primer pair detecting EGFR gene 2236-2253 position disappearance is made up of the upstream primer shown in SEQ ID No:10 and the downstream primer shown in SEQ IDNo:11, and the nucleotide sequence of Taqman-MGB probe is as shown in SEQ ID No:25;

The primer pair detecting EGFR gene 2239-2253 position disappearance is made up of the upstream primer shown in SEQ ID No:12 and the downstream primer shown in SEQ IDNo:13, and the nucleotide sequence of Taqman-MGB probe is as shown in SEQ ID No:27;

The primer pair detecting EGFR gene 2240-2257 position disappearance is made up of the upstream primer shown in SEQ ID No:14 and the downstream primer shown in SEQ IDNo:15, and the nucleotide sequence of Taqman-MGB probe is as shown in SEQ ID No:29;

The primer pair detecting EGFR gene 2582 sudden change is made up of the downstream primer shown in the upstream primer shown in SEQ ID No:16 and SEQ ID No:5, and the nucleotide sequence of Taqman-MGB probe is as shown in SEQ ID No:33;

The primer pair detecting EGFR gene 2573 sudden change is made up of the downstream primer shown in the upstream primer shown in SEQ ID No:17 and SEQ ID No:4, and the nucleotide sequence of Taqman-MGB probe is as shown in SEQ ID No:31.

Preferred, described test kit also comprises the detection primer pair in DNA quality control site and identifies the Taqman-MGB probe of gained amplicon, described primer pair is made up of the downstream primer shown in the upstream primer shown in SEQ ID No:18 and SEQ ID No:19, and the nucleotide sequence of described Taqman-MGB probe is as shown in SEQ ID No:35.

Preferred, described test kit is by PCR damping fluid, dNTPs, primer pair, Taqman-MGB probe, MgCl ₂, Taq DNA polymerase and template DNA composition, during each component reaction, final concentration is:

PCR damping fluid	Final concentration 0.5 ~ 2.5 ×;
		dNTPs	0.1～0.75mM；
Primer pair	Final concentration is respectively 150 ~ 350nM;
		Template DNA	0.01～10ng/μL；
Taq DNA polymerase	0.01～1.0U/μL；
		MgCl ₂	Final concentration is 1.5 ~ 3.5mM;
Taqman-MGB probe	Final concentration is 50 ~ 200nM

Preferred, described PCR damping fluid, dNTPs, primer pair, Taqman-MGB probe, MgCl ₂, Taq DNA polymerase and template DNA final concentration consist of:

PCR buffer Final concentration 1 ×;
	dNTPs0.25mM；
Primer pair final concentration is respectively 250nM;
	Template DNA 0.05 ~ 1.5ng/ μ L;
Taq DNA polymerase 0.01 ~ 0.10U/ μ L;
	MgCl2 final concentration is 2.0 ~ 2.5mM;
Fluorescent probe final concentration is 50nM;

Preferred, described dNTP mixed solution comprises the mixed solution of 10mM dATP, 10mM dCTP, 10mM dTTP, 10mMdGTP; PCR damping fluid comprises Tris ﹒ cl, Repone K, ammonium sulfate, magnesium chloride; At-20 DEG C, pH value is between 8.0-9.0.

Preferred, described template DNA extracts from peripheral blood, frozen section, flesh tissue, paraffin section.

The archaeal dna polymerase concentration 5units/ μ l used in the present invention, reaction substrate is dNTP, ddNTP, is 2-4kb/min 72 DEG C of condition downward-extension speed.In 10 × PCR damping fluid, the concentration of each material is respectively 10-40mM Tris ﹒ cl, 50-200mM Repone K (KCL), 0-5.0mM dithiothreitol (DTT) (DTT), 0-1.0mM Sormetal (EDTA), 0-2.0% (V/V) Nonidet P40 (Nonidet) P-40,0-2.0% (V/V) polysorbas20 (Tween20), 30-70%(v/v) glycerine (glycerol), stablizer (stabilizer): pH7.0-10.0 (20 DEG C).Preferred concentration is 20mM Tris ﹒ cl, 100mM KCL, 1mM DTT, 0.1mM EDTA, 0.5% (V/V) Nonidet P-40,0.5% (V/V) Tween20,50%glycerol(v/v), stablizer, whole pH value is 9.0.DNA profiling is from patient tissue, extract by ordinary method the nucleic acid obtained, comprises DNA and RNA.Wherein patient tissue, comprises the cast of cavity, pathological tissues, and with paraffin mass, paraffin section, frozen section etc. that these tissues are made.

The reaction conditions of test kit of the present invention, as shown in the table:

Preferred reaction conditions:

In the present invention, term " primer " refers to a kind of oligonucleotide, can be natural also can be synthesis, it can as the starting point of induce dna synthesis under certain condition, the primer extension product that synthesis is complementary mutually with nucleic acid chains can be brought out under these conditions, namely under four kinds of different triphosphoric acid thymus nucleic acids and a kind of polymerization agent (i.e. archaeal dna polymerase or reversed transcriptive enzyme) exist, above-mentioned synthesis is carried out at a suitable temperature at a kind of suitable damping fluid.Preferred primer is sub-thread oligodeoxyribonucleotide.The appropriate length of primer depends on the designed use of this primer, but general between 15 ~ 25 Nucleotide, shorter primer molecule needs lower temperature usually, thus forms fully stable hybridization complex with template.Primer need not the exact sequence of reaction template, but must be fully complementary, has hybridized with template and has caused DNA and synthesized.

Design of primers adhere to principled: 1. design and there is specificity in primer application nucleic acid series conserved regions.2. product can not form secondary structure.3. primer length is generally between 15 ~ 30 bases.4. G+C content is between 40% ~ 60%.5. base wants stochastic distribution.6. primer self can not have the complementation of continuous 4 bases.7. the complementation of continuous 4 bases can not be had between primer.8. primer 5 ' end can be modified.9. primer 3 ' end can not be modified.10. primer 3 ' end will avoid the 3rd of codon.The software carrying out design of primers has Primer5, Beacon Designer7.

The method that primer synthesis adopts is phosphoramidite triester method, DNA is fixed on synthesis solid phase carrier completing DNA chain, and the direction of synthesis is held to 5 ' end synthesis by 3 ' of primer to be synthesized, and adjacent Nucleotide is connected by 3 ' → 5 ' phosphodiester bond.

In the present invention, term " fluorescent probe " refers to a kind of oligonucleotide of mark fluorescent signal, is synthetic, it can as combining with specific DNA under certain condition, under the effect that archaeal dna polymerase 5'-3' is exo-acting, send fluorescent signal, accept by quantitative real time PCR Instrument.Preferred probe is the sub-thread oligodeoxyribonucleotide of mark fluorescent signal.The appropriate length of probe depends on the designed use of this probe, general between 10 ~ 25 Nucleotide, although its specificity of shorter probe molecule reduces, but can avoid uncertainty that is complicated because of DNA sequence dna and that cause.The exact sequence of the necessary reaction template of probe.

Probe design adhere to principled: 1. design and there is specificity in probe application nucleic acid series conserved regions.2. product can not form secondary structure.3. probe length is generally between 10 ~ 30 bases.4. G+C content is between 40% ~ 60%.5. base wants stochastic distribution.6. probe self can not have the complementation of continuous 4 bases.7. probe 5' first base can not for G 8. probe 5 ' end add fluorescence radiation group.9. probe 3 ' end adds quenching of fluorescence group.10. in probe sequence, the content of G can not be higher than the content of C.The software carrying out design of primers has Express3.0.

Synthetic method and the primer of probe are similar, just after sequent synthesis, add fluorescence radiation group at the 5' end of oligonucleotide, add quenching of fluorescence group at the 3' end of oligonucleotide.

In the present invention, term " test kit " refers to the mixture for PCR reaction, and it comprises damping fluid (buffer), dNTP mixed solution, primer, fluorescent probe, MgCl2, Taq enzyme needed for reaction.

Beneficial effect of the present invention is: test kit disclosed by the invention, adopts ARMS specific mutagenesis beneficiation technologies to combine with Taqman-MGB probe specific detection technology, without the need to order-checking, can complete the judgement to sample genotype; This test kit is highly sensitive, can reach 1%, and specificity is good simultaneously, and precision is high, and being detected as power is 100%, can detect many middle samples simultaneously, realize high throughput testing; This test kit usage range is wide, can detect the DNA prepared from peripheral blood, frozen section, flesh tissue, paraffin section, provide guidance according to the personalized medicine that the detected result of test kit is tumour.

Accompanying drawing illustrates:

Fig. 1 is that different positions introduces missense mutation result figure (M:Marker; 1: special primer; 2:3 ' holds the 2nd missense mutation; 3:3 ' holds the 3rd missense mutation; 4:3 ' holds the 4th missense mutation).

Fig. 2 is Taqman-MGB probe positive-sense strand and antisense strand result figure.

Fig. 3 is the result figure (M:Marker of different PCR Buffer concentration; 0.5 ×, 1 ×, 1.5 ×, 2 ×, 2.5 × be respectively 0.5 times, 1 times, 1.5 times, 2 times and 2.5 times of PCR damping fluids).

Fig. 4 is different Mg Cl ₂result figure (the M:Marker of concentration; 1.5,2.0,2.5,3.0 and 3.5 represent MgCl respectively ₂concentration 1.5mM, 2.0mM, 2.5mM, 3.0mM and 3.5mM).

Fig. 5 is the result figure (M:Marker of different dNTPs concentration; 0.1,0.25,0.5 and 0.75 represents that dNTPs concentration is respectively 0.1mM, 0.25mM, 0.5mM and 0.75mM respectively).

Fig. 6 is the result figure (M:Marker of different AR MS primer concentration; Primer concentration is respectively 150nM, 250nM, 300nM, 350nM and 400nM).

Fig. 7 is the result figure of different Taqman-MGB concentration and probe concentration.

Fig. 8 test kit susceptibility of the present invention detected result figure.

Embodiment

Below in conjunction with specific embodiment, such scheme is described further.Should be understood that these embodiments are not limited to for illustration of the present invention limit the scope of the invention.The implementation condition adopted in embodiment can do further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in normal experiment.

Embodiment 1

Human EGFR gene is positioned on genome No. 7 karyomit(e), (Genebank:NG_007726.3) of EGFR gene group DNA, and the full-length cDNA of coding EGFR gene is as shown in SEQ ID No:1.Mainly concentrate on exons 19 and exon 21 according to patient's EGFR genetic mutation, EGFR gene exons 19 sequence is as shown in SEQ ID No:2, and EGFR gene exon 21 sequence is as shown in SEQ ID No:3.The mutational site of EGFR gene is as shown in table 1:

Table 1.EGFR mutational site

Build the positive quality control product in EGFR gene hot spot mutation detection kit, be specially the positive reference substance of the disappearance of 2235-2249 position in test kit, 2236-2250 position disappearance, 2236-2253 position disappearance, 2239-2253 position disappearance, 2240-2257 position disappearance, 2582 missense mutation and 2573 missense mutation, reference product when detecting for test kit.

Above-mentioned 7 kinds of positive reference substances, by screening corresponding 7 type mutant DNAs or the corresponding 7 kinds of mutant DNAs of synthetic, being cloned in pGM-T carrier, being built into recombinant plasmid.Then by recombinant plasmid transformation of E. coli DH5 α respectively, after order-checking is defined as recombinant plasmid, recombinant plasmid is extracted, purified rear dilution, obtained positive reference substance, for subsequent use.

Get the positive reference substance after dilution to mix in varing proportions with wild type gene group DNA respectively, obtain wild-type sample (not containing positive reference substance), 1% sudden change sample (ratio of the template of positive reference substance and the template of wild sample is 1:100), 2% sudden change sample (ratio of the template of positive reference substance and the template of wild sample is 2:100), 5% sudden change sample (ratio of the template of positive reference substance and the template of wild sample is 5:100), 10% sudden change sample (ratio of the template of positive reference substance and the template of wild sample is 10:100), 50% sudden change sample (ratio of the template of positive reference substance and the template of wild sample is 50:100), 100% sudden change sample (not containing wild type gene group DNA), make the OD260/OD280 of sample after mixing between 1.8 ~ 2.0, make Positive mutants sample, for subsequent use.

ARMS beneficiation technologies combines with Taqman-MGB probe techniques by the present invention, realizes the detection to mutational site.Because missense mutation is not mated with wild type gene group DNA only having 3 ' last bit base, non-specific amplification may be there is, in order to avoid ARMS primer pair is combined with wild type gene group DNA, primer is designed respectively according to mutational site, the last bit base making missense mutation site be positioned at 3 ' of upstream primer to hold, and manually introduce base mismatch at the 3' end of upstream primer to 5 ' end the 2nd, 3 and 4 difference, concrete primer is as shown in table 2, and the downstream primer of 2573 missense mutation is: 5 '-aaacaatacagctagtgggaaggc-3 ' (SEQ ID No:4); The downstream primer of 2582 missense mutation is 5 '-gaaggcagcctggtccctggtgtcagg-3 ' (SEQ ID No:5).Be that template carries out PCR reaction by the primer of design and corresponding positive reference substance, PCR reaction conditions is: 95 DEG C of denaturations 5 minutes, a circulation; 40 amplification cycles, 95 DEG C are out of shape 20 seconds, anneal and extend 40 seconds for 60 DEG C; Last 40 DEG C of coolings 15 seconds, result as shown in Figure 1.As shown in Figure 1, hold 2-4 position to introduce base mismatch at the 3' end of ARMS upstream primer to 5 ' and all can amplify object band, but best base mismatch position of introducing is at the 2nd.

The ARMS upstream primer of table 2. different positions missense mutation

According to above-mentioned experimental result and EGFR mutational site, design detects the ARMS primer pair in EGFR gene exons 19 and EGFR gene exon 21 mutational site, detects its essence of ARMS primer pair of EGFR gene exons 19 for detecting 2235-2249 position disappearance, 2236-2250 position disappearance, 2236-2253 position disappearance, 2239-2253 position disappearance and 2240-2257 position disappearance; Its essence of ARMS primer pair detecting exon 21 is detection 2582 missense mutation and 2573 missense mutation.Because the mutation type of EGFR gene exons 19 is disappearance, therefore detect primer with the design of the mutant nucleotide sequence of disappearance, and make the upstream primer of detection primer across absent region, downstream primer design is in the intron region in EGFR gene exons 19 downstream; Because the mutation type of EGFR gene exon 21 is missense mutation, therefore according to the sequences Design ARMS primer pair of missense mutation, the last bit base making missense mutation site be positioned at 3 ' of upstream primer to hold, to be positioned at 3 ' end to 5 ' the 2nd the Nucleotide people held is missense mutation simultaneously, obtains ARMS primer pair as shown in table 3.The primer in design dna quality control site simultaneously.

Table 3. detects the ARMS primer pair in EGFR mutational site

Utilize Express3.0 software design Taqman-MGB probe, the positive-sense strand (complementary with amplified production antisense strand) and antisense strand (complementary with amplified production positive-sense strand) of ARMS primer design Sense probes and antisense probe respectively, specifically as shown in table 4, take positive reference substance as template, nucleotide sequence shown in table 2 is ARMS primer, nucleotide sequence shown in table 3 is respectively Taqman-MGB probe, carry out quantitative fluorescent PCR, PCR reaction conditions is: 95 DEG C of denaturations 5 minutes, a circulation; 40 amplification cycles, 95 DEG C are out of shape 20 seconds, anneal and extend 40 seconds for 60 DEG C; Last 40 DEG C of coolings 15 seconds.Sense probes and antisense probe amplification curve are compared, result as shown in Figure 2.As shown in Figure 2, (identify the positive-sense strand in amplified production mutational site) when probe is antisense probe, its specificity, amplification efficiency are best.

Table 4. Sense probes and antisense probe nucleotide sequence

Therefore, according to the above results, with the antisense probe in table 4 for Taqman-MGB probe, contain fluorescent reporter group at 5 ' end of nucleotide sequence Taqman-MGB probe simultaneously, 3 ' end, containing fluorescent quenching group, also has minor groove binding molecule (MGB) at 3 ' end.

Embodiment 2

PCR is carried out respectively with the primer in table 3 and 7 positive control template, in PCR reaction PCR Buffer concentration gradient be respectively 0.5 ×, 1 ×, 1.5 ×, 2 × and 2.5 ×, all the other constituent concentrations are identical, and (in 20 μ L systems, the final concentration of each component is respectively 0.25mM dNTP, 2.5mM MgCl ₂, 1U/ reaction dna polysaccharase, 250nM primer, 30ng template DNA and surplus be water).PCR reaction conditions is: 95 DEG C of denaturations 5 minutes, a circulation; 40 amplification cycles, 95 DEG C are out of shape 20 seconds, anneal and extend 40 seconds for 60 DEG C; Last 40 DEG C of coolings 15 seconds.Pcr amplification product is by electroresis appraisal, and result as shown in Figure 3.As shown in Figure 3, be by amplified band in 0.5 × ~ 2.5 × scope in PCR Buffer concentration gradient, when PCRBuffer concentration is its result the best of 1 × time.

Embodiment 3

MgCl is carried out respectively with the primer in table 3 and 7 positive control template ₂the PCR that concentration is different, all the other component conditions identical (in 20 μ L systems, the final concentration of each composition is 1 × PCR Buffer, 0.25mM dNTP, 1U/ reaction dna polysaccharase, 250nM primer, 30ng template DNA, and surplus is water), MgCl ₂concentration is respectively 1.5mM, 2.0mM, 2.5mM, 3.0mM and 3.5mM.PCR reaction conditions is identical with embodiment 2, and amplified production carries out electroresis appraisal, and result as shown in Figure 4.As shown in Figure 4, at MgCl ₂concentration all has object band to increase within the scope of 1.5mM ~ 3.5mM, works as MgCl ₂concentration is best results when 2.0-2.5mM.

Embodiment 4

Carry out the pcr amplification of different dNTPs concentration respectively with the primer in table 3 and 7 positive control template, other conditions are fixed, and (in 20 μ l systems, each composition final concentration is 1 × PCR Buffer, 2.5mM MgCl ₂, 1U/ reaction dna polysaccharase, 250nM primer, 30ng template DNA, surplus be water), dNTPs concentration is respectively 0.1mM, 0.25mM, 0.5mM and 0.75mM.PCR reaction conditions is identical with embodiment 2, and amplified production carries out electroresis appraisal, and result as shown in Figure 5.As shown in Figure 5, be all have object product amplification between 0.25mM ~ 0.75mM in dNTPs concentration, when dNTP concentration is 0.25mM, its result is best.

Embodiment 5

Carry out the pcr amplification of different primers concentration respectively with the primer in table 3 and 7 positive control template, other conditions are fixed, and (in 20 μ L systems, each composition final concentration is 1 × PCR Buffer, 2.5mM MgCl ₂, 0.25mM dNTP, 1U/ reaction dna polysaccharase, 30ng template DNA, surplus be water), primer concentration is respectively 150nM, 250nM, 300nM and 350nM.PCR reaction conditions is identical with embodiment 2, and amplified production carries out electroresis appraisal, and result as shown in Figure 6.As shown in Figure 6, primer concentration all can amplify object band within the scope of 150-350nM, but when primer concentration is 250nM its best results.

Embodiment 6

With the primer in table 3 and 7 positive reference substances for template carries out the quantitative fluorescent PCR of different antisense probe concentration, other conditions are fixed, and (in 20 μ l systems, the final concentration of each material is 1 × PCR Buffer, 2.5mM MgCl ₂, 0.25mM dNTP, 1U/ reaction dna polysaccharase, 250nM primer, 30ng template DNA, surplus be water), concentration and probe concentration is respectively 50nM, 100nM, 150nM and 200nM, and quantitative fluorescent PCR reaction conditions is: 95 DEG C of denaturations 5 minutes, a circulation; 40 amplification cycles, 95 DEG C are out of shape 20 seconds, anneal and extend 40 seconds for 60 DEG C; Last 40 DEG C of coolings 15 seconds, react and carry out on quantitative real time PCR Instrument.According to the best working concentration of amplification curve qualification probe, for 2235-2249 position disappearance amplification curve, as shown in Figure 7.Can show that the impact of concentration and probe concentration on PCR is less according to fluorescent quantitation amplification curve and all can fluorescent signal be detected at 50-200nM, and best results when concentration and probe concentration is 50nM.

Embodiment 7

Carry out the pcr amplification of different hot start Taq polymerase with the primer in table 3 and 7 positive control template, other conditions are fixed, and (in 20 μ L systems, the final concentration of each material is 1 × PCR Buffer, 2.5mM MgCl ₂, 0.25mM dNTP, 1U/ reaction dna polysaccharase, 250nM primer, 30ng template DNA, surplus be water).The present embodiment uses HotStarTaq enzyme, FastStartTaq enzyme, Taq CE DNA Polymerase, KAPA2G rapid hot start archaeal dna polymerase respectively, and pcr amplification condition is with embodiment 3, and Enzyme assay result is as shown in table 5.

As shown in Table 5, the activity of HotStarTaq enzyme and FastStartTaq enzyme is the highest, and KAPA2G rapid hot start archaeal dna polymerase enzymic activity is poor.

From embodiment 1 ~ 7, detect in the test kit in EGFR genetic mutation site, each material final concentration is in use as follows:

PCR damping fluid	Final concentration 0.5 ~ 2.5 ×;
		dNTPs	0.1～0.75mM；
ARMS primer pair	Upstream and downstream primer final concentration is respectively 150 ~ 350nM;
		Template DNA	0.01～10ng/μL；
Taq DNA polymerase	0.01～1.0U/μL；
		MgCl ₂	Final concentration is 1.5 ~ 3.5mM;
Taman-MGB probe	Final concentration is 50 ~ 200nM

Wherein can comprise the whole ARMS primer pairs in table 3 in test kit, also can be the part primer in table 3, also primer can be selected as required during detection, as: detect in EGFR gene exons 19 whether there is the deletion mutantion of 2235-2249 position, only need select the primer and corresponding Taqman-MGB probe that detect 2235-2249 position disappearance.As: in uncertain sample to be tested, whether EGFR gene undergos mutation, the type of also uncertain sudden change and site, then detected respectively by all primers in test kit.Conveniently detect, the present invention is selective temperature wide accommodation when designing primer, and react under the following conditions and all can realize specific amplified, condition is: 95 DEG C of denaturation 2-15 minute; 95 DEG C of sex change 10-30 seconds, 50-65 DEG C, 10-60 realize second, anneal and extend, this step circulation 35-50 time; Finally be cooled to 40 DEG C.On the other hand, all ARMS primer pairs in table 2 can detect under same reaction conditions simultaneously, therefore can realize high throughput testing.

Embodiment 8

DNA is extracted respectively from peripheral blood, frozen section, flesh tissue, paraffin section, for detection template DNA, other testing conditions select the experiment condition of the optimization of embodiment 1 ~ 7, detected result shows, the template DNA extracted in frozen section, flesh tissue, paraffin section all can detect, and otherness is little.And the template DNA concentration extracted from peripheral blood (serum/plasma) is lower, Taqman-ARMS experimental result is unstable.

Embodiment 9

With the wild-type sample of preparation in embodiment 1,1% sudden change sample, 2% sudden change sample, 5% sudden change sample, 10% sudden change sample, 50% sudden change sample, 100% sudden change sample for template, primer in table 2, by the preferred condition of embodiment 1 ~ 8, (final concentration is 1 × PCR Buffer, 2.5mM MgCl to other conditions ₂, 0.25mM dNTP, 1U/ reaction dna polysaccharase, 250nM primer, 30ng template DNA, Taqman-MGB probe 50nM, surplus be water) carry out quantitative fluorescent PCR, the condition of quantitative fluorescent PCR is with embodiment 6, and result is as shown in Figure 8.As shown in Figure 8, test kit detection sensitivity disclosed by the invention can reach 1%.

Embodiment 10

Choose cancerous lung tissue paraffin section 40 example, extract DNA as template, detect with the ARMS primer pair of detection 2582 missense mutation and the Taqman-MGB probe of correspondence, testing conditions is with embodiment 9; The DNA that 40 routine cancerous lung tissues extract is checked order simultaneously.

Taqman-ARMS detection display, in the paraffin section sample of 40 routine cancerous lung tissues, test kit detects that 16 routine pattern detection have sudden change somatotype, and order-checking has 15 examples for sudden change somatotype, and order-checking is the sample of sudden change somatotype, it is also sudden change somatotype that test kit detects, and it is 95% that two kinds of methods detect concordance rate.It can thus be appreciated that be detected as power 100% based on the test kit of Taqman-ARMS technology, positive detection goes out rate and reaches order-checking and compare consistence 100%, and the accurate sensitivity detecting sudden change exceedes order-checking.

Application Example 1

Get paraffin section 30 example of patients with lung cancer, sample is by BJ Union Hospital, and the attached First Hospital of Zhejiang University, Xijing hospital provides, and extracts DNA as template.Use instrument: LlightCyclerTM480 quantitative fluorescent PCR instrument is analyzed, by the ARMS primer pair in table 2 and corresponding Taqman-MGB probe in detecting, quantitative fluorescent PCR condition is with embodiment 9.Fluorescent quantitative PCR result shows, in 30 routine patients with lung cancer, detect 3 routine 2235-2249 position disappearances, 2 routine 2236-2250 position disappearances, 2 routine 2240-2257 position disappearances, 4 example 2582 missense mutation, detect 12 routine EFGR transgenation patients altogether, sudden change recall rate reaches 37%, the data consistent reported with report domestic and foreign literature.

Application Example 2

Get the frozen section sample of the 30 routine patients with lung cancer identical with Application Example 1, extract DNA as template.Use instrument: LlightCyclerTM480 quantitative fluorescent PCR instrument is analyzed, by the ARMS primer pair in table 2 and corresponding Taqman-MGB probe in detecting, quantitative fluorescent PCR condition is with embodiment 9.Fluorescent quantitative PCR result shows, in 30 routine patients with lung cancer, the patient of 3 routine 2235-2249 position disappearances is detected in frozen section sample paraffin section, the patient of 2 routine 2236-2250 position disappearances, the patient of 2 routine 2240-2257 position disappearances, the patient of 4 examples, 2582 missense mutation, detect 12 example sudden change patients altogether, sudden change recall rate reaches 37%, consistent with the detected result of paraffin section, meets the data of domestic and foreign literature report simultaneously.

Application Example 3

Get the fresh case sample of the 30 routine patients with lung cancer identical with Application Example 1, extract DNA as template.Use instrument: LlightCyclerTM480 quantitative fluorescent PCR instrument is analyzed, by the ARMS primer pair in table 2 and corresponding Taqman-MGB probe in detecting, quantitative fluorescent PCR condition is with embodiment 9.Fluorescent quantitative PCR result shows, in 30 routine patients with lung cancer patients, detect the patient of 3 routine 2235-2249 position disappearances, the patient of 2 routine 2236-2250 position disappearances, the patient of 2 routine 2240-2257 position disappearances, the patient of 4 examples, 2582 missense mutation, detect 12 example sudden change patients altogether, sudden change recall rate reaches 37%.Consistent with Application Example 1 and Application Example 2.

What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by referring to the preferred embodiments of the present invention, invention has been described, but those of ordinary skill in the art is to be understood that, various change can be made to it in the form and details, and not depart from the present invention that appended claims limits.

<110> Suzhou Micro Diag Biomedicine Co., Ltd.

<120> detects the test kit in EGFR genetic mutation site

<130>

<160> 35

<210> 1

<211> 3633

<212> DNA

<213> homo sapiens ( homo sapiens) EGFR gene CDS

<400> 1

atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60

gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag 120

ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg 180

gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag 240

accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct 300

ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca 360

gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta 420

caggaaatcc tgcatggcgc cgtgcggttc agcaacaacc ctgccctgtg caacgtggag 480

agcatccagt ggcgggacat agtcagcagt gactttctca gcaacatgtc gatggacttc 540

cagaaccacc tgggcagctg ccaaaagtgt gatccaagct gtcccaatgg gagctgctgg 600

ggtgcaggag aggagaactg ccagaaactg accaaaatca tctgtgccca gcagtgctcc 660

gggcgctgcc gtggcaagtc ccccagtgac tgctgccaca accagtgtgc tgcaggctgc 720

acaggccccc gggagagcga ctgcctggtc tgccgcaaat tccgagacga agccacgtgc 780

aaggacacct gccccccact catgctctac aaccccacca cgtaccagat ggatgtgaac 840

cccgagggca aatacagctt tggtgccacc tgcgtgaaga agtgtccccg taattatgtg 900

gtgacagatc acggctcgtg cgtccgagcc tgtggggccg acagctatga gatggaggaa 960

gacggcgtcc gcaagtgtaa gaagtgcgaa gggccttgcc gcaaagtgtg taacggaata 1020

ggtattggtg aatttaaaga ctcactctcc ataaatgcta cgaatattaa acacttcaaa 1080

aactgcacct ccatcagtgg cgatctccac atcctgccgg tggcatttag gggtgactcc 1140

ttcacacata ctcctcctct ggatccacag gaactggata ttctgaaaac cgtaaaggaa 1200

atcacagggt ttttgctgat tcaggcttgg cctgaaaaca ggacggacct ccatgccttt 1260

gagaacctag aaatcatacg cggcaggacc aagcaacatg gtcagttttc tcttgcagtc 1320

gtcagcctga acataacatc cttgggatta cgctccctca aggagataag tgatggagat 1380

gtgataattt caggaaacaa aaatttgtgc tatgcaaata caataaactg gaaaaaactg 1440

tttgggacct ccggtcagaa aaccaaaatt ataagcaaca gaggtgaaaa cagctgcaag 1500

gccacaggcc aggtctgcca tgccttgtgc tcccccgagg gctgctgggg cccggagccc 1560

agggactgcg tctcttgccg gaatgtcagc cgaggcaggg aatgcgtgga caagtgcaac 1620

cttctggagg gtgagccaag ggagtttgtg gagaactctg agtgcataca gtgccaccca 1680

gagtgcctgc ctcaggccat gaacatcacc tgcacaggac ggggaccaga caactgtatc 1740

cagtgtgccc actacattga cggcccccac tgcgtcaaga cctgcccggc aggagtcatg 1800

ggagaaaaca acaccctggt ctggaagtac gcagacgccg gccatgtgtg ccacctgtgc 1860

catccaaact gcacctacgg atgcactggg ccaggtcttg aaggctgtcc aacgaatggg 1920

cctaagatcc cgtccatcgc cactgggatg gtgggggccc tcctcttgct gctggtggtg 1980

gccctgggga tcggcctctt catgcgaagg cgccacatcg ttcggaagcg cacgctgcgg 2040

aggctgctgc aggagaggga gcttgtggag cctcttacac ccagtggaga agctcccaac 2100

caagctctct tgaggatctt gaaggaaact gaattcaaaa agatcaaagt gctgggctcc 2160

ggtgcgttcg gcacggtgta taagggactc tggatcccag aaggtgagaa agttaaaatt 2220

cccgtcgcta tcaaggaatt aagagaagca acatctccga aagccaacaa ggaaatcctc 2280

gatgaagcct acgtgatggc cagcgtggac aacccccacg tgtgccgcct gctgggcatc 2340

tgcctcacct ccaccgtgca gctcatcacg cagctcatgc ccttcggctg cctcctggac 2400

tatgtccggg aacacaaaga caatattggc tcccagtacc tgctcaactg gtgtgtgcag 2460

atcgcaaagg gcatgaacta cttggaggac cgtcgcttgg tgcaccgcga cctggcagcc 2520

aggaacgtac tggtgaaaac accgcagcat gtcaagatca cagattttgg gctggccaaa 2580

ctgctgggtg cggaagagaa agaataccat gcagaaggag gcaaagtgcc tatcaagtgg 2640

atggcattgg aatcaatttt acacagaatc tatacccacc agagtgatgt ctggagctac 2700

ggggtgaccg tttgggagtt gatgaccttt ggatccaagc catatgacgg aatccctgcc 2760

agcgagatct cctccatcct ggagaaagga gaacgcctcc ctcagccacc catatgtacc 2820

atcgatgtct acatgatcat ggtcaagtgc tggatgatag acgcagatag tcgcccaaag 2880

ttccgtgagt tgatcatcga attctccaaa atggcccgag acccccagcg ctaccttgtc 2940

attcaggggg atgaaagaat gcatttgcca agtcctacag actccaactt ctaccgtgcc 3000

ctgatggatg aagaagacat ggacgacgtg gtggatgccg acgagtacct catcccacag 3060

cagggcttct tcagcagccc ctccacgtca cggactcccc tcctgagctc tctgagtgca 3120

accagcaaca attccaccgt ggcttgcatt gatagaaatg ggctgcaaag ctgtcccatc 3180

aaggaagaca gcttcttgca gcgatacagc tcagacccca caggcgcctt gactgaggac 3240

agcatagacg acaccttcct cccagtgcct gaatacataa accagtccgt tcccaaaagg 3300

cccgctggct ctgtgcagaa tcctgtctat cacaatcagc ctctgaaccc cgcgcccagc 3360

agagacccac actaccagga cccccacagc actgcagtgg gcaaccccga gtatctcaac 3420

actgtccagc ccacctgtgt caacagcaca ttcgacagcc ctgcccactg ggcccagaaa 3480

ggcagccacc aaattagcct ggacaaccct gactaccagc aggacttctt tcccaaggaa 3540

gccaagccaa atggcatctt taagggctcc acagctgaaa atgcagaata cctaagggtc 3600

gcgccacaaa gcagtgaatt tattggagca tga 3633

<210> 2

<211> 99

<212> DNA

<213> homo sapiens ( homo sapiens) EGFR gene the 19th exon

<400> 2

ggactctgga tcccagaagg tgagaaagtt aaaattcccg tcgctatcaa ggaattaaga 60

gaagcaacat ctccgaaagc caacaaggaa atcctcgat 99

<210> 3

<211> 156

<212> DNA

<213> homo sapiens ( homo sapiens) EGFR gene the 21st exon

<400> 3

ggcatgaact acttggagga ccgtcgcttg gtgcaccgcg acctggcagc caggaacgta 60

ctggtgaaaa caccgcagca tgtcaagatc acagattttg ggctggccaa actgctgggt 120

gcggaagaga aagaatacca tgcagaagga ggcaaa 156

<210> 4

<211> 24

<212> artificial sequence

The downstream primer of <213> 2573 missense mutation

<400> 4

aaacaataca gctagtggga aggc 24

<210> 5

<211> 27

<212> artificial sequence

The downstream primer of <213> 2582 missense mutation

<400> 5

gaaggcagcc tggtccctgg tgtcagg 27

<210> 6

<211> 29

<212> artificial sequence

<213> detects 2235-2249 position disappearance upstream primer

<400> 6

ttaaaattcc cgtcgctatc aaaacatct 29

<210> 7

<211> 21

<212> artificial sequence

<213> detects 2235-2249 position disappearance downstream primer

<400> 7

cccacacagc aaagcagaaa c 21

<210> 8

<211> 29

<212> artificial sequence

<213> detects 2236-2250 position disappearance upstream primer

<400> 8

aaaattcccg tcgctatcaa gacatctcc 29

<210> 9

<211> 21

<212> artificial sequence

<213> detects 2236-2250 position disappearance downstream primer

<400> 9

cccacacagc aaagcagaaa c 21

<210> 10

<211> 28

<212> artificial sequence

<213> detects 2236-2253 position disappearance upstream primer

<400> 10

aaaattcccg tcgctatcaa gtctccga 28

<210> 11

<211> 21

<212> artificial sequence

<213> detects 2236-2253 position disappearance downstream primer

<400> 11

cccacacagc aaagcagaaa c 21

<210> 12

<211> 28

<212> artificial sequence

<213> detects 2239-2253 position disappearance upstream primer

<400> 12

tcccgtcgct atcaaggaat ctccgaaa 28

<210> 13

<211> 21

<212> artificial sequence

<213> detects 2239-2253 position disappearance downstream primer

<400> 13

cccacacagc aaagcagaaa c 21

<210> 14

<211> 27

<212> artificial sequence

<213> detects 2240-2257 position disappearance upstream primer

<400> 14

tcccgtcgct atcaaggaat cgaaagc 27

<210> 15

<211> 21

<212> artificial sequence

<213> detects 2240-2257 position disappearance downstream primer

<400> 15

cccacacagc aaagcagaaa c 21

<210> 16

<211> 22

<212> artificial sequence

<213> detects 2582 missense mutation upstream primers

<400> 16

tcaagatcac agattttggc cg 22

<210> 17

<211> 21

<212> artificial sequence

<213> detects 2573 missense mutation upstream primers

<400> 17

agattttggg ctggccaacc a 21

<210> 18

<211> 28

<212> artificial sequence

<213> DNA quality control site upstream primer

<400> 18

tctctctctg tcatagggac tctggatc 28

<210> 19

<211> 21

<212> artificial sequence

<213> DNA quality control sites downstream primer

<400> 19

cccacacagc aaagcagaaa c 21

<210> 20

<211> 15

<212> artificial sequence

<213> 2235-2249 position disappearance Sense probes

<400> 20

gctatcaaaa catct 15

<210> 21

<211> 15

<212> artificial sequence

<213> 2235-2249 position disappearance antisense probe

<400> 21

agatgttttg atagc 15

<210> 22

<211> 16

<212> artificial sequence

<213> 2236-2250 position disappearance Sense probes

<400> 22

ctatcaagac atctcc 16

<210> 23

<211> 16

<212> artificial sequence

<213> 2236-2250 position disappearance antisense probe

<400> 23

ggagatgtct tgatag 16

<210> 24

<211> 17

<212> artificial sequence

<213> 2236-2253 position disappearance Sense probes

<400> 24

cgctatcaag tctccga 17

<210> 25

<211> 17

<212> artificial sequence

<213> 2236-2253 position disappearance antisense probe

<400> 25

tcggagactt gatagcg 17

<210> 26

<211> 18

<212> artificial sequence

<213> 2239-2253 position disappearance Sense probes

<400> 26

atcaaggaat ctccgaaa 18

<210> 27

<211> 18

<212> artificial sequence

<213> 2239-2253 position disappearance antisense probe

<400> 27

tttcggagat tccttgat 18

<210> 28

<211> 15

<212> artificial sequence

<213> 2240-2257 position disappearance Sense probes

<400> 28

caaggaatcg aaagc 15

<210> 29

<211> 15

<212> artificial sequence

<213> 2240-2257 position disappearance antisense probe

<400> 29

gctttcgatt ccttg 15

<210> 30

<211> 14

<212> artificial sequence

<213> 2573 missense mutation Sense probes

<400> 30

ttgggggggc caaa 14

<210> 31

<211> 14

<212> artificial sequence

<213> 2573 missense mutation antisense probes

<400> 31

tttggccccc ccaa 14

<210> 32

<211> 14

<212> artificial sequence

<213> 2582 missense mutation Sense probes

<400> 32

caaagagctg ggtg 14

<210> 33

<211> 14

<212> artificial sequence

<213> 2582 missense mutation antisense probes

<400> 33

cacccagctc tttg 14

<210> 34

<211> 21

<212> artificial sequence

<213> DNA quality control site Sense probes

<400> 34

tgagaaagtt aaaattcccg t 21

<210> 35

<211> 21

<212> artificial sequence

<213> DNA quality control site antisense probe

<400> 35

acgggaattt taactttctc a 21

Claims

1. detect the test kit in EGFR genetic mutation site, it is characterized in that: described test kit comprises the Taqman-MGB probe of ARMS primer pair and specific recognition 2582 sudden change detecting EGFR gene 2582 sudden change;

Described ARMS primer pair is made up of the downstream primer shown in the upstream primer shown in SEQ ID No:16 and SEQ ID No:5;

The Taqman probe of amplified production positive-sense strand 2582 sudden change of described Taqman-MGB probe specific recognition ARMS primer, 3 ' end of described probe is combined with minor groove binding molecule, and nucleotide sequence is as shown in SEQ ID No:33.

2. detect the test kit in EGFR genetic mutation site according to claim 1, it is characterized in that: described test kit also comprises the detection primer pair in DNA quality control site and identifies the Taqman-MGB probe of gained amplified production, described primer pair is made up of the downstream primer shown in the upstream primer shown in SEQ ID No:18 and SEQ ID No:19, and the nucleotide sequence of described Taqman-MGB probe is as shown in SEQ ID No:35.

3. the test kit in described detection EGFR genetic mutation site according to claim 2, is characterized in that: described test kit is by PCR damping fluid, dNTPs, primer pair, Taqman-MGB probe, MgCl ₂, Taq DNA polymerase and template DNA composition, during each component reaction, final concentration is:

PCR buffer Final concentration 0.5 ~ 2.5 ×;

dNTPs 0. 1～0.75mM；

Primer pair final concentration is respectively 150 ~ 350nM;

Template DNA 0.01 ~ 10ng/ μ L;

Taq DNA polymerase 0.01 ~ 1.0U/ μ L;

MgCl ₂final concentration is 1.5 ~ 3.5mM;

Taqman-MGB probe final concentration is 50 ~ 200nM.

4. detect the test kit in EGFR genetic mutation site according to claim 3, it is characterized in that: described PCR damping fluid, dNTPs, primer pair, Taqman-MGB probe, MgCl ₂, Taq DNA polymerase and template DNA final concentration consist of:

PCR buffer Final concentration 1 ×;

dNTPs 0.25mM；

Primer pair final concentration is respectively 250nM;

Template DNA 0.05 ~ 1.5ng/ μ L;

Taq DNA polymerase 0.01 ~ 1.0U/ μ L;

MgCl ₂final concentration is 2.0 ~ 2.5mM;

Taqman-MGB probe final concentration is 50nM.

5. detect the PCR reaction kit of EGFR genetic mutation according to claim 4, it is characterized in that: described dNTP mixed solution comprises the mixed solution of 10 mM dATP, 10 mM dCTP, 10 mM dTTP, 10 mM dGTP; Described PCR damping fluid comprises Tris ﹒ cl, Repone K, ammonium sulfate, magnesium chloride; At-20 DEG C, pH value is between 8.0-9.0.

6. detect the PCR reaction kit of EGFR genetic mutation according to claim 4, it is characterized in that: described template DNA extracts from peripheral blood, frozen section, flesh tissue, paraffin section.