CN104846108B

CN104846108B - Detect primer, kit and its PCR method in C-KIT gene D816V mutational sites

Info

Publication number: CN104846108B
Application number: CN201510290519.2A
Authority: CN
Inventors: 高劲松; 张英杰; 魏欣芳; 李星颐; 魏潇; 魏奇
Original assignee: SHENYANG EUGENOM BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: SHENYANG EUGENOM BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2015-05-29
Filing date: 2015-05-29
Publication date: 2018-04-27
Anticipated expiration: 2035-05-29
Also published as: CN104846108A

Abstract

The invention discloses a kind of primer, kit and its PCR method in detection C KIT gene D816V mutational sites, including：The anti-sense primer that wild type specific forward primer, saltant type specific forward primer and wild type specific forward primer are shared with saltant type specific forward primer；And the wild type specific forward primer has SEQ No.17 sequences, the saltant type specific forward primer has SEQ No.14 sequences, and the shared anti-sense primer has SEQ No.16 sequences；Kit has the advantages that detection is simple, quick, accurate, cheap, and strong instrument is provided for scientific research and clinic C KIT gene D816V site partings and gene mutation analysis.

Description

Detect primer, kit and its PCR method in C-KIT gene D816V mutational sites

Technical field

The present invention relates to molecular biology genetic test field, specifically provides a kind of drawing for detection C-KIT gene mutations Thing, kit and its PCR method, the quick detection for C-KIT gene D816V mutational sites.

Background technology

C-KIT genes belong to proto-oncogene, and the KIT acceptors of coding belong to III type tyrosine kinase.This receptor is with matching somebody with somebody Body --- stem cell factor（SCF）With reference to rear, downstream signal is activated by forming dimer, including Ras/Raf/MAPK paths, Akt/PI3K paths etc., finally activate intracytoplasmic transcription factor, adjust gene expression, control cell growth and propagation.

Under normal circumstances, KIT acceptors activate under the participation effect of stem cell factor.If C-KIT genes are undergone mutation, KIT receptor activations are not required ligand SCF to participate in, so as to cause the continuous proliferation of tumour cell, and cause apoptotic signal path It is out of control.Research finds that the mutation of C-KIT genes can cause the generation of malignant tumour, including acute myelocytic leukemia, intestines and stomach Mesenchymoma and carcinoma of testis etc..

Wherein gastrointestinal stromal tumor（Gastrointestinal Stromal Tumor, GIST）It is that alimentary canal is most common Mesenchymal tissue source property tumour.The Kit albumen of the GIST tumor cells expression C-KIT gene codes of the overwhelming majority（CD117）, and GIST is commonly present C-KIT gene mutations, and mutation causes the activation of KIT albumen to be not required ligand SCF to participate in regard to tumour can be stimulated thin The continuous proliferation of born of the same parents and anti-apoptotic signal it is out of control, so that tumour occur.C-KIT gene mutation rates are about 80-90% in GIST, Mutant form is various, including replaces, lacks and be inserted into repetition etc..Wherein positioned at the prominent of exons 1 1Lys550-Val560 sections Become most commonly seen（Account for 70-80%）, repeatedly mutation accounts for 5-10% for the 6 bases insertion of 9 Ala502-Tyr503 sections of extron.

Imatinib（Imatinib）, also known as Glivec（Gleevec）It is small molecule tyrosine kinase inhibitors, it can be selected Suppress KIT, BCR-ABL and PDGFR to selecting property.Imatinib is incorporated into the ATP of tyrosine kinase functional areas in KIT albumen endochylemas Binding site, blocks transfer of the phosphate group from ATP to substrate tyrosine residue, so as to suppress cell Proliferation and recover cell to wither Die program.At present, Imatinib is used to treat the KIT positives, is unable to surgery excision and/or metastatic malignant GIST, it is clinical Curative effect is exciting.But and not all GIST patient can obtain good efficacy after Imatinib is treated, clinical research shows The effect of catastrophe of C-KIT genes is with Imatinib molecular targeted therapies in GIST is closely related.There are C-KIT extrons Patient's curative effect of 11 mutation is best, and patient's curative effect that extron 9 is mutated is taken second place, and GIST patient's curative effect without gene mutation is most Difference.In addition, the patient being mutated to extron 9, dosage was brought up to 800mg/ days by 400mg/ days can improve curative effect.

Another targeted drug Sutent in 2006（Sunitinib, also known as sotan Sutent）Obtain U.S. FDA approval For the drug resistant intractable GIST patients of Imatinib.It is similar to Imatinib, the clinical efficacy of Sunitinib and gene mutation Site is related.Unlike, it is best to 9 mutation person's effect of C-KIT extrons, and the effect of 11 mutation person of extron is paid no attention to Think.

American cancer integrated network（NCCN）《Soft tissue sarcoma's clinical therapeutic guideline》Middle proposition, GIST patient receive targeting Before drug therapy, C-KIT detection in Gene Mutation should be first carried out, decides whether to use targeted drug as clinical according to testing result Remedy measures.Detection C-KIT gene mutations have important for instructing rationally being treated using molecular targeted agents for GIST patient Reference value.

It is common to have direct sequencing at present to the detection of gene mutation and gene pleiomorphism；Gene chip hybridization method；PCR- RFLP methods；Pcr amplification product capillary electrophoresis analysis method；PCR-SSCP；PCR high-resolution fusion curve analytical technologies；PCR- Taqman MGB（Minor Groove Binder）Sonde method；AS-PCR methods；Cast-PCR methods etc..These methods are more or less Also high there are instrument price, operation difficulty is big, and there are certain false negative and false positive, and testing cost is high, and clinical popularity is low, The shortcomings of clinical samples cannot be detected on a large scale at the same time.

Such as：1）Direct sequencing is the goldstandard of mutation analysis, can find known and unknown mutation site, but the method detects The sensitivity of gene mutation is 20%（That is the mutator DNA profiling number of target gene accounts for the 20% of wild type DNA profiling number）.Together When also there are complicated, cycle length, analyze speed is slow, often needs the time of 2 days, and the method is open pipe operation, is increased Add the possibility of pollution, be not suitable for the detection to extensive sample, while also need to the instrument of costliness, exist and be not easy reality in basic unit The shortcomings of applying.

2）Regular-PCR-RFLP method and technologies are easy, cheap, the test in laboratory of suitable a small amount of sample, but RFLP is only The mutation for having restriction enzyme site can be detected, no restriction enzyme site cannot detect, time-consuming and laborious, and also there are PCR product pollution to cause false sun The risk of property, is shown in [2010 Jun 1 of Mol Diagn Ther.;14(3):163-9, United States Patent 20120135406]。

3）Chip technology has the characteristics that high throughput, micromation, automation compared with traditional instrument detection method, is applicable in In full genome mutated scanning, it is not suitable for the mutational site detection of individual gene, and precision is low, it is expensive.

4）PCR high-resolution fusion curve analytical technologies, the catastrophe of energy high throughput detection gene, reagent cost is relatively low, But because its fluorescence signal comes from dyestuff, specificity is affected, and detecting instrument is the fluorescent PCR instrument of upgrade version, and price is high High, popularization is limited, sees [2012 Nov 12 of Clin Chim ACqa.;413(21-22):1781-5; United States Patent 20110045479]。

5）PCR-Taqman MGB sonde methods are adapted to known mutations site, it is often necessary to two probes, and Taqman MGB The synthesis price of probe is several times of general T aqman probes, sees [2010 Aug of J Clin Microbiol.;48(8): 2909-15；United States Patent 20090311679], and cannot detect the content in sample it is less (>=1, 1 in 000) allele or mutational site.

6）Although PCR-SSCP methods are simple, which is open detecting system, be easy to cause the pollution of PCR product, It is time-consuming and laborious and operating procedure is more.

7）Allele-specific primers PCR amplification method (AS-PCR), this method are current detection gene mutation or SNP Most simple and quick method（Wu D Y, Ugozzoli L, Pal B K, Wallace R B., Proc Natl Acad Sci USA 1989; 86:2757-2760), its principle is the base mispairing of primer 3' terminal bases and template, the effect of its PCR Rate will decline 10³-10^6.6(Chen, X., and Sullivan, P F, The Pharmacogeonomics again Journal 2003, 3, 77-96).Although the principle of this method is simple, the primer of mispairing, can still occur non-specificity Amplification, amplification situation regard type (Ayyadevara S, the Thaden J related to the base sequence around detection site of mutation J, Shmookler Reis R J., Anal Biochem 2000; 284:11-18), also with equipotential base present in sample The influence of dependent variable.In order to increase the specificity of AS-PCR, many scientists have done many effort.Largely it is demonstrated experimentally that should For method it is crucial that design and 3' terminal mutations site base complementrity or two special primers of mispairing, design of primers is bad, The intersection for causing high background is expanded, higher false positive can be caused.Although many people make great efforts to overcome this drawback, such as report TaqMAMA methods, in the upper specificity for introducing mutating alkali yl, reaction being increased really of 3 ' penultimates or the 3rd, But still false positive can not be completely eliminated, and in the judgement of homozygote and heterozygote, lack standard, it may appear that chaotic feelings Condition, is shown in [2008 Nov of J Virol Methods.;153(2):156-62;Genomics. 2004 Feb;83(2):311- 20].Simple AS-PCR, generally use PCR carry out electrophoresis again after reaction, from there is reactionless band to carry out judging result, this Although the instrument that kind of method need not be expensive, electrophoretic procedures, the opportunities for contamination of PCR, and time and effort consuming are added.In spite of People improves this method, and using fluorescent quantitative PCR technique, but what is obtained is not " all or none " formula as a result, always having non- The generation of specific reaction, i.e., same primer pair wild type and saltant type site can all expand, simply its obtained Ct （Cycle threshold）It is different, therefore just introduce the concept of Δ Ct, i.e. Δ Ct=wild Ct- mutation Ct, but calculate Complexity, such as wants Δ Ct values to be judged to saltant type homozygote more than homozygote Ct values, Δ Ct values are less than heterozygote Ct values, are judged to heterozygosis Son, the introducing of Δ Ct values not only increase operating procedure, can also cause confusion, because the established standards of Δ Ct values can not accurately be determined Position, different samples and different detecting instruments can all have different numerals, and very big difficulty is brought to clinical practice, actual to answer With being still limited, see [Chinese patent CN101235415, CN101565742A].

8）LIFE companies of the U.S. are referred to as Cast-PCR using a kind of method of MGB closing probes（ Competitive allele specific Taqman PCR）, the site do not detected is closed with MGB probes, is then drawn with allele specific The method testing goal site of thing quantitative fluorescent PCR, although this improves the specificity of detection, due to adding a MGB more Probe is closed, cost certainly will be increased, how much interference can be brought to reaction efficiency, see [2012 Jun of Exp Mol Pathol.;92 (3):275-80； United States Patent 20100221717；CN102428190A].Someone uses lock nucleic acid (LNA) (Plant Method 2007,3：2) or modification base (Anal Biochem.2005,340：PCR 287-294) Primer, can cause AS-PCR detection sensitivities to improve.However, these approaches increases the overall cost of analysis, and need to be to anti- It should carry out depth optimization.

At present, a kind of C-KIT gene mutation detection liquid-phase chips of domestic Guangzhou Yishan Biotechnology Co., Ltd.'s application C-KIT mutation are analyzed using liquid-phase chip technology（101984070 A of patent No. CN）；Xiamen Ai De biological medicines section A kind of probe, primer and detection kit for being used to detect C-KIT gene mutations of skill Co., Ltd application uses real-time fluorescence The detection C-KIT mutation of PCR methods（103114133 A of patent No. CN）.

Chip technology has the characteristics that high throughput, micromation, automation compared with traditional instrument detection method, is suitable for Full genome mutated scanning, is not suitable for the mutational site detection of individual gene, and precision is low, expensive.And common fluorescence Quantitative PCR technique designs the most key site-specific primer, also only carries out, does not have according to the rule of design of primers The screening objective indicator of one good special primer, the most key is that their result judges, still obscures, does not have " all or none " Concept, it is so non-specific the shortcomings that still can not overcome.

Therefore, simple accurately detection how is carried out to C-KIT gene D816V mutational sites, it is urgently to be resolved hurrily to become people The problem of.

The content of the invention

In consideration of it, the present invention provides a kind of primer, kit and its PCR in detection C-KIT gene D816V mutational sites Method, at least to solve result interpretation complexity existing for conventional kit, detecting instrument price height, operation difficulty is big, and there are one Determine false negative and false positive, testing cost is high, and clinical popularity is low, it is impossible at the same extensive detection clinical samples etc. one or Multiple problems.

Scheme provided by the invention is specially：A kind of primer in detection C-KIT gene D816V mutational sites, its feature exist In the primer includes：Wild type specific forward primer, saltant type specific forward primer and wild type specific upstream draw The anti-sense primer that thing is shared with saltant type specific forward primer；

And the wild type specific forward primer has SEQ No.17 sequences, the saltant type specific forward primer With SEQ No.14 sequences, the shared anti-sense primer has SEQ No.16 sequences.

One aspect of the present invention additionally provides a kind of reagent in detection C-KIT gene D816V mutational sites, it is characterised in that： The reagent contains above-mentioned primer.

Another aspect of the present invention additionally provides a kind of kit in detection C-KIT gene D816V mutational sites, its feature exists In：The kit also contains above-mentioned primer.

It is preferred that the probe being used cooperatively with the primer is further included in the kit, wherein, the probe is Taqman Probe, has SEQ No.15 sequences.

Further preferably, the kit further includes PCR reaction tubes, dNTPs and Taq enzyme mixed liquor, PCR buffer solutions, inspection Survey the spy of the sense primer of house-keeping gene GAPDH, the anti-sense primer of detection house-keeping gene GAPDH, detection house-keeping gene GAPDH Pin, positive quality control product and blank control；

The sense primer of the detection house-keeping gene GAPDH has SEQ No.19 sequences；The detection house-keeping gene The anti-sense primer of GAPDH has SEQ No.20 sequences；The probe of the detection house-keeping gene GAPDH has SEQ No.21 sequences Row.

Further preferably, the saltant type specific forward primer, the shared anti-sense primer, the probe, described Detect the sense primer of house-keeping gene GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene The probe of GAPDH is coated in T-shaped PCR reaction tubes in advance；And with the wild type specific forward primer, it is described share Anti-sense primer, the probe, the sense primer of the detection house-keeping gene GAPDH, under the detection house-keeping gene GAPDH The probe of trip primer and the detection house-keeping gene GAPDH are coated in A type PCR reaction tubes in advance.

Further preferably, saltant type specific forward primer in the T-shaped PCR reaction tubes, the shared anti-sense primer, The probe, the sense primer of the detection house-keeping gene GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and institute The concentration and probe concentration for stating detection house-keeping gene GAPDH is respectively 50nM-2um, 50nM-2uM, 50nM-400nM, 1pm-20pm, 1pm- 20pm、0.05pm-5pm；And wild type specificity sense primer, the shared downstream are drawn in the A types PCR reaction tubes Thing, the probe, the sense primer of the detection house-keeping gene GAPDH, the detection house-keeping gene GAPDH anti-sense primer and The concentration and probe concentration of the detection house-keeping gene GAPDH be respectively 50nM-2um, 50nM-2uM, 50nM-400nM, 1pm-20pm, 1pm-20pm、0.05pm-5pm。

Still more preferably, saltant type specific forward primer, the shared downstream are drawn in the T-shaped PCR reaction tubes Thing, the probe, the sense primer of the detection house-keeping gene GAPDH, the detection house-keeping gene GAPDH anti-sense primer and The concentration and probe concentration of the detection house-keeping gene GAPDH is respectively 500nM, 500nM, 100nM, 5pm, 5pm, 2.5pm；It is and described Wild type specificity sense primer, the shared anti-sense primer, the probe, detection house keeper's base in A type PCR reaction tubes Because of the spy of the sense primer of GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene GAPDH Pin concentration is respectively 500nM, 500nM, 100nM, 5pm, 5pm, 2.5pm.

Present invention also offers above-mentioned primer and the PCR method of kit, it is characterised in that：PCR reactions are expanded by two steps Cyclic program carries out, and the period of first step amplification is less than the period of second step amplification, the annealing of the first step amplification Temperature is higher than the annealing temperature of second step amplification.

It is preferred that the condition of the PCR reactions is：37 DEG C of 2min, 95 DEG C of 2min pre-degenerations；The first step expands, 95 DEG C 5s is denatured, 55 DEG C ~ 68 DEG C annealing 32s, are circulated 10 ~ 15 times；Second step, 95 DEG C of denaturation 5s, 50 DEG C ~ 65 DEG C annealing, prolong 32s is stretched, is circulated 30 ~ 50 times, collects fluorescence signal；Further preferably, the condition of the PCR reactions is：37 DEG C of 2min, 95 DEG C 2min pre-degenerations；The first step expands, and 95 DEG C of denaturation 5s, 63 DEG C of annealing 32s, are circulated 15 times；Second step, 95 DEG C of denaturation 5s, 60 DEG C of annealing, extension 32s, circulates 40 times, collects fluorescence signal.

The primer and its kit in detection C-KIT gene D816V mutational sites provided by the invention, can greatly increase Position gene-specific primer PCR sector divides the ability in not iso-allele site, makes the non-specific intersection amplification between different loci Possibility be preferably minimized, can accomplish the amplification of real " all or none " formula, not only have good specificity, it may have very good Sensitivity, the genotype distribution situation in 1ng genomic DNAs can be detected.

The primer and its kit in detection C-KIT gene D816V mutational sites provided by the invention, have the following advantages：

1）Result " all or none " is judged by being truly realized testing result the artificial change on primer sequence, significantly The difficulty of result interpretation is simplified, reduces the possibility of error, is provided convenience for scientific research and clinical practice.

2）The mixing of Taq enzyme and dNTPs, has ensured the stability of dNTPs, it is subjected to examining for multiple multigelation Test.

3）The buffer solution that can be used directly pre-assigned in advance, user are more convenient.

4）Primer and probe is coated in PCR reaction tubes in advance, avoids the possibility that site mismatch occurs in operator, and Also save a large amount of operating times.

5）Primer and kit have specific good in the present invention, and the high sensitivity of detection, detection speed is fast, whole process It can be completed when 1 is small in 20 minutes.

6）Only probe is not closed such as without other, is reduced cost with a routine Taqman probe in kit.

7）The kit has the advantages that detection is simple, quick, accurate, inexpensive.

Brief description of the drawings

PCR amplification curve map when Fig. 1 is design wild type and mutant primers；

Fig. 2 is the PCR amplification curve map of detection mutated genes sequence；

Fig. 3 is the PCR amplification curve map of detection wildtype gene sequence；

Fig. 4 is the PCR amplification curve map of house-keeping gene GAPDH.

Embodiment

The present invention is further expalined with specific case below, but the protection model being not intended to limit the invention Enclose.

Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art, Product used is purchased in market.

Defined below is the explanation of relational language in the present invention：

" allele " generally refers to DNA section, in the same, physical on homologue, controls relativity One pair of genes.In some cases, allele can correspond to the replacement of the mononucleotide on specific physics locus. In other situations, allele can correspond to (single or multiple) insertions of nucleotide or missing.

" allele-specific primers " refers to complementary with the sequence of target alleles, can extend completion in PCR Special PCR reactions.Allele-specific primers can be designed to detect in target sequence as little as 1 nucleotide difference ten The different primer of dtex, the primer can include allele-specific nucleotide part, 3 ' end target specificity parts and tail.

" MGB groups " refers to minor groove binding.When being conjugated with the 3 ' of oligonucleotides ends, MGB groups can be used as not Extendible enclosure portion plays a role.MGB is the molecule that can be incorporated into the ditch of double-stranded DNA, generally use DPI3（It is closed Into method referring to U.S. Patent number 6,084,102；With 6,727,356).

" detection probe " refers to：Indicate any one in the multi-signal transduction molecule of amplification.For example, SYBR Green All it is detection probe with other DNA binding dyes.Some detection probes can be sequence-specific, such as Taqman probes (U.S. Patent number 5,538,848), a variety of stem ring molecular beacons (U.S. Patent number 6,103,476 and 5,925,517 and Tyagi and Kramer, Nature Biotechnology, 1996,14：303-308), acaulescence or Linear Beacon (WO99/ 21881), PNA Molecular Beacons TM (U.S. Patent number 6,355,421 and 6,593,091), linear PNA beacons (Kubista et al., 2001, SPIE4264：53-58), non-FRET probes (U.S. Patent number 6,150,097), Sunrise/ Amplifluor probes (U.S. Patent number 6,548,250), stem ring and double-strand Scorpion TM probes (Solinas et al., 2001, NucleicAcidsResearch29：E96 and U.S. Patent number 6,589,743), the ring probe (U.S. Patent number heaved 6,590,091), false knot probe (U.S. Patent number 6,589,250), cyclicon (U.S. Patent number 6,383,752), MGB Eclipse TM probes (Epoch Biosciences), hairpin probe (U.S. Patent number 6,596,490), peptide nucleic acid (PNA) are visited Pin.Detection probe can include fluorescent reporter molecule, for example, 6- Fluoresceincarboxylic acids (6-FAM) or tetrachlorofluorescein (TET), and Quencher molecule part, such as tetramethylrhodamin (TAMRA), Black Hole Quenche RS (Biosearch), Iowa Black (IDT), QSY quenchers (Molecular Probes) and Dabsyl and Dabcel sulfonate/carboxylate quencher (Epoch)。

" shared specific primer " refers to the primer with allele-specific primers pairing in PCR reacts, it can be same The allele-specific primers pairing of different loci uses.

" polymerase of heat endurance " refers to heat endurance or heat resistance enzyme, refers mainly to archaeal dna polymerase, which exists During PCR amplification, it will not inactivate at an elevated temperature.

When " Tm " or " melting temperature " of oligonucleotides refers to 50% molecule and complementary sequence hybridization in section of DNA Temperature.Tm values can be calculated (see Maniatis, T. et al. using well known formula：Molecular cloning, Cold Spring Harbor, N.Y.：1982).

" sensitivity " is to refer to be detected the minimum (copy number) of template.

" specificity " refers to the ability for distinguishing matching template and mispairing template.Specificity is often expressed as Δ Ct=Ct mistakes Matched with-Ct.

" selectivity " refer to AS-PCR measure can be used for minority (typically be mutated) allele in measure mixture and Degree without the interference from most (often wild type) allele.Selectivity is often expressed as ratio or percentage.Example Such as, the measure that 1 mutagenesis template can be detected in the case of there are 100 wild-type templates is referred to as having 1: 100 or 1% Selectivity.

" Ct " value refers to cycle threshold, represents period when PCR amplification curve is changed into exponential increase flex point from baseline.

" Delta Ct " or " when Δ Ct " refers to that signal passes through fixed threshold, the circulation between two different samples or reaction Number difference.Δ Ct is the period difference between two different samples or reaction when reaching exponential amplification.Δ Ct can be used for identifying Match the specificity between primer and mismatched primers.

The present invention relates to the primer and kit in quick detection C-KIT gene D816V mutational sites.Specifically, this hair Bright be based on the basis of Past-PCR (Perfect allele-specific Taqman PCR) method, is successfully applied to C- Detect in KIT gene D816V mutational sites.So-called Past-PCR is the high specific AS-PCR side by detection gene SNP or mutation The Fluorescence PCR assay of method and Taqman probes is combined into, it is only with a Taqman probe, under most of the cases, this Probe is conventional Taqman probes, because being rich in the special sequences of AT bases only in portion gene, using Taqman-MGB or The probes such as LNA, in addition without any extra closing probe or reporter probe, Past-PCR methods by AS-PCR and Taqman fluorescent PCR two methods perfect adaptations, and make to have obtained the performance of extreme the advantages of both, it is in particular in AS-PCR In method, in the case where not increasing any cost, non-specificity is successfully eliminated by design of primers and unique verification method Amplification so that result judges to show " complete and nothing " mode, that is, having just has, and does not just have so that the judgement of result it is simple and Accurately, this is the improvement to past AS-PCR methods maximum, and AS-PCR methods are effectively made up for what This is what people generally disapprove of, from And the advantages of AS-PCR methods, has been performed to ultimate attainment, it is the attainable tidemark of this method institute.It is well known that Taqman is glimmering Light PCR has seal detection without reacting the processing after finishing, and not only saves the time, and reduce PCR product pollution Risk, second of probe identification target sequence ensured the specificity and reliability of detection, it may also be used for gene is quantified The advantages that detecting, having reached criterion for clinical use.Past-PCR methods have AS-PCR methods and Taqman fluorescence PCR methods Two-fold advantage, and when being that both approaches are combined together, the attainable most simplified composition of detection component institute, therefore, we Call it as perfect allele-specific Taqman PCR.

Based on Past-PCR methods, the kit for detecting gene C-KIT gene D816V mutational sites has been invented, its It basic composition is：(a) allele-specific primers of high special；(b) fluorescent detection probe；(c) and allele specific Property primer pairing another specific primer.

Wherein, the primer in detection C-KIT gene D816V mutational sites provided by the invention, including：On wild type specificity What trip primer, saltant type specific forward primer and wild type specific forward primer were shared with saltant type specific forward primer Anti-sense primer；

Detection reagent and kit containing above-mentioned primer are additionally provided at the same time, is dashed forward with solving detection C-KIT genes in the past Become big there are operation difficulty, there are certain false negative and false positive, and testing cost is high, and clinical popularity is low, it is impossible at the same it is big Scale detects the problems such as clinical samples, realizes and detects simple, quick, accurate, cheap desirable, is scientific research and clinic C- KIT gene D816V site partings and gene mutation analysis provide strong instrument.

It is preferred that the probe being used cooperatively with the primer is further included in the kit, wherein, the probe is Taqman Probe, has SEQ No.15 sequences, a probe is used only in whole kit, does not close probe etc. without other, reduces The cost of kit.

The sense primer of the detection house-keeping gene GAPDH has SEQ No.19 sequences；The detection house-keeping gene The anti-sense primer of GAPDH has SEQ No.20 sequences；The probe of the detection house-keeping gene GAPDH has SEQ No.21 sequences Row；Wherein, dNTPs and Taq enzyme are mixed, can ensures the stability of dNTPs, it is subjected to examining for multiple multigelation Test；Sense primer, anti-sense primer and the probe of detection house-keeping gene GAPDH is for use as internal reference, to go out when preventing detection Existing false negative；Positive quality control product is T-shaped（Or A types）Human gene group DNA；Blank control is sterilizing ultra-pure water.

Further preferably, the saltant type specific forward primer, the shared anti-sense primer, the probe, described Detect the sense primer of house-keeping gene GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene The probe of GAPDH is coated in T-shaped PCR reaction tubes in advance；And with the wild type specific forward primer, it is described share Anti-sense primer, the probe, the sense primer of the detection house-keeping gene GAPDH, under the detection house-keeping gene GAPDH The probe of trip primer and the detection house-keeping gene GAPDH are coated in A type PCR reaction tubes in advance, are used by optimal reaction Amount is coated in PCR reaction tubes in advance, six oligonucleotides is actually coated with each reaction tube, i.e., one is directed to allele A kind of specific forward primer, a probe（Usually Taqman probes）, a shared specific downstream primer, an inspection Survey house-keeping gene GAPDH sense primers, a detection house-keeping gene GAPDH anti-sense primer and a detection house-keeping gene GAPDH Probe.So in order to detect a different gene pleiomorphism in site, often need the PCR being coated with respectively more than two pipes or two pipes anti- Ying Guan, their middle probes, share specific downstream primer, sense primer, anti-sense primer and the probe of detection house-keeping gene GAPDH It is the same, different is only the identification other specific forward primer of allele different shaped, and so coating avoids behaviour in advance There is the possibility of site mismatch in author, and has saved a large amount of operating times.

Further preferably, saltant type specific forward primer in the T-shaped PCR reaction tubes, the shared anti-sense primer, The probe, the sense primer of the detection house-keeping gene GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and institute The concentration and probe concentration for stating detection house-keeping gene GAPDH is respectively 50nM-2um, 50nM-2uM, 50nM-400nM, 1pm-20pm, 1pm- 20pm、0.05pm-5pm；And wild type specificity sense primer, the shared downstream are drawn in the A types PCR reaction tubes Thing, the probe, the sense primer of the detection house-keeping gene GAPDH, the detection house-keeping gene GAPDH anti-sense primer and The concentration and probe concentration of the detection house-keeping gene GAPDH be respectively 50nM-2um, 50nM-2uM, 50nM-400nM, 1pm-20pm, 1pm-20pm、0.05pm-5pm.It is optimal to be, it is saltant type specific forward primer in the T-shaped PCR reaction tubes, described shared Anti-sense primer, the probe, the sense primer of the detection house-keeping gene GAPDH, the downstream of the detection house-keeping gene GAPDH The concentration and probe concentration of primer and the detection house-keeping gene GAPDH are respectively 500nM, 500nM, 100nM, 5pm, 5pm, 2.5pm；With And wild type specificity sense primer, the shared anti-sense primer, the probe, the detection in the A types PCR reaction tubes The sense primer of house-keeping gene GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene The concentration and probe concentration of GAPDH is respectively 500nM, 500nM, 100nM, 5pm, 5pm, 2.5pm.

Due to introducing the base of mispairing in allele-specific primers, decline its efficiency in reaction, no Same primer declines degree difference, usually to increase effects when 10 to 20 circulations can be only achieved no mispairing, ensure to detect Sensitivity, preferably PCR reaction by two step amplification cycles programs carry out, and the first step amplification period be less than second step amplification Period, the annealing temperature of first step amplification is higher than the annealing temperature of second step amplification, and first step amplification use is higher Annealing temperature, second step expand use compared with low temperature thermal oxidation, it is therefore an objective to specificity when increasing the first step amplification during primer annealing, So as to ensure the purpose of Past-PCR detection high degree of specificity；Specifically the condition of preferably described PCR reactions is：37℃ 2min, 95 DEG C of 2min pre-degenerations；The first step expands, and 95 DEG C of denaturation 5s, 55 DEG C ~ 68 DEG C annealing 32s, are circulated 10 ~ 15 times； Second step, 95 DEG C of denaturation 5s, 50 DEG C ~ 65 DEG C annealing, extension 32s, circulate 30 ~ 50 times, collect fluorescence signal；It is more highly preferred to PCR reaction condition be：37 DEG C of 2min, 95 DEG C of 2min pre-degenerations；The first step expands, 95 DEG C of denaturation 5s, 63 DEG C of annealing 32s, is circulated 15 times；Second step, 95 DEG C of denaturation 5s, 60 DEG C of annealing, extension 32s, circulate 40 times, collect fluorescence signal.

Specifically detection process is：

1）Detected sample is respectively put into and is coated with wild type special primer, shared anti-sense primer respectively in advance, visits Pin, the sense primer of detection house-keeping gene GAPDH, the anti-sense primer of detection house-keeping gene GAPDH, detection house-keeping gene GAPDH Probe and saltant type special primer, shared anti-sense primer, probe, the sense primer of detection house-keeping gene GAPDH, detection In the anti-sense primer of house-keeping gene GAPDH, two groups of PCR reaction tubes of probe of detection house-keeping gene GAPDH, and add PCR bufferings Liquid, dNTPs and Taq enzyme mixed liquor, sterilizing ultra-pure water and genomic DNA, cover tightly tube cover, are carried out on fluorescence quantitative PCR instrument anti- Should, and collect fluorescence signal；

2）If detection sample only has the amplification curve that saltant type reaction tube has logarithm to increase in FAM passages, while VIC leads to There is the amplification curve that logarithm increases in road, and can determine that sample to be T-shaped, i.e. saltant type homozygote；

If detection sample only has the amplification curve that wild type reaction tube has logarithm to increase in FAM passages, while VIC passages There is the amplification curve that logarithm increases, can determine that sample is A types, i.e. wild-type homozygote；

, may if detecting the amplification curve that sample saltant type and wild type reaction tube increase in FAM passages without logarithm Sample or operation should extract sample DNA again and be tested there are problem, or sample DNA concentration are too low.

Each component introduction in detection process is explained in detail below：

（One）Allele-specific primers：

The allele-specific nucleotide partial complementarity of allele-specific primers is in an allele of gene Site, but another allele site of the gene is not complementary to.Typically, the equipotential base of allele-specific primers Because specific nucleotide acid moieties are located at the last bit base at 3 ' ends of allele-specific primers.In some cases, equipotential base Because specific nucleotide acid moieties may be alternatively located at other base positions of the end of allele-specific primers 3 '.

In some cases, allele-specific primers is in addition to 3 ' termini-complementaries are varied from allele site, We are often required to introduce the base of mispairing in the other positions of primer, such as hold penultimate or the 3rd introducing alkali in primer 3 ' Base mispairing, or second and the continuous mispairing of the 3rd；For different SNP or mutational site, penultimate is held in primer 3 ' Or on the basis of the 3rd introducing base mispairing, also it can increase base mispairing in other sites of primer to meet atopic Requirement, further away from 3 ' ends, it is more to increase the base number of mispairing, at 5 ' ends up at most.The length of allele-specific primers Its T of degree Main Basiss_mValue determines, typically its T_mValue is about 5-20 DEG C lower than the annealing temperature of PCR cycle.

The allele-specific sense primer of the corresponding gene pleiomorphism of two detections, except its base of site of 3 ' end detections Different outer, remaining sequence is as identical as possible, the T of two primers_mValue is also as consistent as possible.

Low T_mAllele-specific primers (ASP) have the specificity of higher.Under normal circumstances, allele-specific Primer is short oligonucleotide, its length is about 15-30, its T_mIt it is about 40 DEG C to 60 DEG C, than the PCR used in amplification procedure Annealing/elongating temperature of cycling condition is about 5 DEG C to 20 DEG C low.

Since the design and screening of allele-specific primers are particularly significant, good primer needs to draw from numerous to be selected Selected in thing, therefore we establish a method screened, the specific screening process of its allele-specific primers is as follows：

(1) peripheral primer and PCR amplification are designed：First in gene mutation point to be checked or the upstream and downstream of SNP site, design one To PCR amplification primer, this is then used to carry out conventional PCR to target gene and react primer, wherein expanding fragment length can be Tens to many kilobases pair, and preferred length is 200 to 500 base-pairs；

(2) clone PCR products：Common carrier T matter is arrived in the PCR product that will be amplified, the method cloned using AT, restructuring In grain, recombinant plasmid is obtained, wherein obtained recombinant plasmid can carry out sequence verification, so that it is guaranteed that the correctness of PCR product；

(3) recombinant mutant is built：The information provided according to bioinformatics, using molecule clone technology, by mutant Sequence, build into recombinant plasmid obtained above, the recombinant mutant of structure be subjected to sequence verification sequence, so that it is guaranteed that Its correctness；

(4) design and screen specific primer on the mutated site：Two allele-specific primerses are designed, primer 3 ' ends are complementary with mutant nucleotide sequence, and 3 ' ends of another primer are complementary with normal sequence；Then drawn respectively with allele-specific Thing and the common anti-sense primer pairing of corresponding Standard PCR, it is glimmering using the above-mentioned recombinant mutant built as reaction template, utilization Fluorescent Quantitative PCR instrument, to reaction system, is screened；Wherein, reaction can use fluorescent dye, such as SYBR GREEN, can also use Specific Taqman fluorescence probe, is screened, it has according to added sample size for the standard of 0.1 microgram complete genome DNA The screening content of body is as follows：

（a）Set PCR reaction conditions：First set PCR reaction annealing temperature it is completely the same, actual temp for 55 DEG C~ 68 DEG C, PCR cycle number is 40 times, and the enzyme and buffer system that PCR reacts immobilize after optimizing, the setting of this reaction condition Be for convenience and meanwhile detect several genes be mutated；

（b）Specificity screening：The primer of saltant type is used using the recombinant plasmid of wild type as template, the primer filtered out its Ct is more than 40；

The definite reason of wherein cycle threshold is as follows, and in wild-type template non-specific amplification occurs for mutant primers than wild In wild-type template 19 circulations more than specific amplification occur for raw type primer, and when being detected according to clinical practice, added sample size is The standard of 0.1 microgram complete genome DNA, we pass through following equation, copy number=(amount of DNA ng × 6.022 × 10²³)/(length bp×10⁹× 660), the wherein length of complete genome DNA is 3,000,000,000 pairs of bases, therefore the copy of 0.1 microgram complete genome DNA Number is：3.09×10⁴, it is reflected in the Ct of quantitative fluorescent PCR（Quantitative cycle threshold）About 21, so we obtain second Screening index, i.e., with the primer of saltant type, when the recombinant plasmid of wild type is template, the Ct of gained should be more than 40, can It is not in false positive to ensure detection, both amplification efficiency differences circulate for 40-21=19, i.e., amplification efficiency differs 19 More than circulation, the objective indicator of allele-specific primers specificity quality will be judged as us.It is understood that work as primer When 3 ' ends are with template mispairing, extension efficiency will decline 10³-10^6.6Times, it is converted into reaction cycle number and then declines about 13 to 27 and follows Ring, here it is the theoretical foundation that we can filter out preferable primer, because without primer screening, 19-13=6 circulation certainly will False positive is produced, the specificity of the primer is just bad, and 19-27=- 8, it is impossible to produces false positive, the scope for there are 8 periods Primer is screened for us.According to this requirement, we design a series of allele-specific primerses, from length, Set about from being artificially introduced close to 3 ' ends on base mispairing, plus the anti-sense primer of its pairing, using the wild type built and dash forward Modification recombinant plasmid, carries out the intersection screening of quantitative fluorescent PCR reaction respectively, i.e., when with the template of mutant primer and wild type into During row amplification, with to be used more than 19 periods with the reaction of wild-type template with wild primers, vice versa, then this The primer of sample just possesses the specificity of height.

（c）Sensitiveness is screened：The specific primer filtered out is made into sensitivity tests with recombination mutation type plasmid, will be examined The specific primer that sensitivity is surveyed less than 10-1000 copy screens, and is required mutant-specific primers.

By the screening of above several respects, obtained allele-specific primers will be provided with high degree of specificity and height Sensitive preferable primer.

（Two）Detection probe：

In some cases, the T of detection probe_mValue is about 60 DEG C to 70 DEG C, and most preferably 65 DEG C, probe length is according to it T_mValue determines.It is available in spite of a variety of various forms of probes, Taqman probes (Applied Biosystems, Foster City) it is often preferred.In some special cases, such as AT too high levels in detection sequence, it can use and improve T_mThe probe of the types such as the Taqman-MGB of value.

Shared specific primer, in some cases, the T of shared specific primer_mValue is about 50 DEG C to 70 DEG C, Most preferably 60 DEG C, its length is by its T_mValue determines, generally between 15-25 bases.

（Three）Other compositions：

The archaeal dna polymerase that the present invention uses is Taq enzyme, and its mutant, derivative or fragment or other heat-resisting Archaeal dna polymerase, in some cases, can also use Hotstart Taq enzymes, with the special and high efficiency of increase reaction.

The primer and kit that are there is provided for C-KIT gene D816V mutational sites provided in the present invention, mainly The shortcomings that for allele-specific primers PCR amplification method, using molecule clone technology, structure contains gene to be checked respectively in advance The recombinant plasmid of wild type and saltant type, using this recombinant plasmid as positive template, screening determines specifically to dash forward with 3' ends respectively Become two special primers of site or wild site base complementrity, once the specific primer sequences filtered out, will be that we detect The key element of system.The said goods of the present invention, available for the fluorescence quantitative PCR instrument of any model, reagent cost is the same as nowadays Quantitative fluorescent PCR reagent on the market is suitable, therefore preferably overcomes other and use allele-specific primers PCR amplification The deficiency of method.

The advantage of the invention is that：The kit has easy to operate, and cost is low, and it is simple easy as a result to judge, spirit Sensitivity is high, and the detection gene mutation sensitivity of this kit can reach 1%（That is the mutator DNA template numbers of target gene account for The 1% of wild type DNA profiling number）；And the sensitivity that gene mutation is detected in direct Sequencing is 20%（That is the mutation base of target gene Because DNA template numbers account for the 20% of wild type DNA template numbers）；Taqman probe techniques ensured kit to be detected as stopped pipe anti- Should, the generation of false positive is effectively avoided, it detects specificity and is also fully guaranteed, and detection is quick and convenient, entirely Detection process only has 80 minutes, and direct Sequencing then needs the time of 2 days, and is open pipe operation, PCR product pollution Possibility increases.

PCR in the present invention（Past-PCR）Implementation steps：

1. the structure of positive plasmid

Upstream and downstream in C-KIT gene D816V mutational sites to be detected designs one couple of PCR primers, its amplified fragments Length can be template with gDNA in the range of 500bp, using conventional PCR method, amplify this fragment, cloned by AT Mode, which is cloned into the plasmid of sequencing, usually use Invitrogen pCR2.1 Topo carrier T kits, By specification is operated to carry out, it is also possible to the carrier T kit of other producers, or even can use the carrier T plasmid oneself prepared.Obtain Positive colony, through sequence verification, it was demonstrated that the correctness of sequence, then using Stratagene companies Quickchange try Agent box, designs another genotyping primer of corresponding site, by the method for Quickchange, obtains the other sun of the saltant type Property clone, operation by specification carries out, and obtained positive plasmid must be all allowed for access in next step through sequence verification to be correct Use.Taqman quantifies plasmid, and as template to verify the sensitivity of given determination method, linear dynamic range, special Property.

2. the design and screening of allele-specific primers (ASP)

In the termini-complementary of primer 3 ' in corresponding gene mutation base position, and in the end of primer 3 ' penultimate or Three introducing base mispairings, or second and the continuous mispairing of the 3rd；For different SNP or mutational site, held in primer 3 ' On the basis of penultimate or the 3rd introducing base mispairing, also it can increase base mispairing in other sites of primer to meet The requirement of atopic, further away from 3 ' ends, it is more to increase the base number of mispairing, at 5 ' ends up to most allele specifics Property primer.

The screening of ASP is using the wild type and saltant type recombinant plasmid built, carries out quantitative fluorescent PCR reaction respectively Intersection screening, i.e., when being expanded with the template of mutant primer and wild type, and with the same wild-type template of wild primers Reaction to use more than 19 periods, vice versa, then such primer just possesses the specificity of height.It will filter out Specific primer make sensitivity tests with corresponding recombinant plasmid, detection sensitivity is less than the 10-1000 primer copied Reach the sensitivity requirement of detection, the primer for meeting above-mentioned two condition is exactly the ASP primers that we select, and can be carried out next The detection of step.

3. amplifing reagent prepares and adds tested sample

（1）Take out each constituent from kit, room temperature slowly melt PCR buffer solutions, dNTPs and Taq enzyme mixed liquor, Positive quality control product, blank control, it is reverse to shake up.Each reaction tube need to add PCR buffer solutions, Taq enzyme（dNTPs）, sterilizing it is ultrapure Water and DNA sample.Tube cover is covered tightly, PCR amplification area is transferred to after brief centrifugation.

（2）It is recommended that each PCR reactions are carried out at the same time sample, positive quality control product, the analysis of blank control, positive quality control product Loading methods with blank control are the same as sample Adding Way.

4. reaction condition

(scope of reaction final volume can be in 10-50 μ l, most preferably in 20-25 μ l reaction volumes) is included in each reaction tube PCR buffer solutions（10mM Tris-HCl pH8.3,50mM KCl, 0.1%Triton X-100,2.0mM MgCl₂）, 10- 100ng DNA or 1, the Plasmid DNA of 000,000 copy, mono- common general specificity T aqman of 50nM-400nM are visited Pin, and the general reverse specific downstream primer that a 50nM-2uM is common, and 50nM-2uM difference allele-specifics draw Thing, the sense primer of 1pm-20pm detection house-keeping genes GAPDH, the anti-sense primer of 1pm-20pm detection house-keeping genes GAPDH, The probe of 0.05pm-5pm detection house-keeping genes GAPDH, along with 1.5 units of Taq polymerase, 200 μM of dNTPs.

1）, cycling condition set

To make fluorescence curve more beautiful, it is proposed that collect fluorescence signal since second step.

2）, instrument sense channel selection

Fluorescence signal acquisition temperature is set to 60 DEG C, and specific method to set up refer to corresponding instrument operation instructions.

3）, detection type setting：Blank control is set to " NTC ", and positive quality control product and sample to be checked are set to " Unknown ".

5. interpretation of result

If detection sample only has the amplification curve that saltant type reaction tube has logarithm to increase in FAM passages, while VIC passages The amplification curve for having logarithm to increase, can determine that sample to be T-shaped, i.e. saltant type homozygote；It is anti-that if detection sample only has wild type Should pipe FAM passages have logarithm increase amplification curve, while VIC passages have logarithm increase amplification curve, can determine that sample For A types, i.e. wild-type homozygote；Due to this method the result is that " all or none ", as a result easily judges, which pipe has reaction, which One pipe is exactly positive.

Specific embodiment

Below by taking the situation detected to C-KIT gene D816V mutational sites as an example, specifically the present invention is made further detailed Explanation.

Embodiment 1：The preparation of wild type and saltant type positive plasmid for C-KIT gene D816V mutational sites

C-KIT encoded K IT acceptors participate in adjusting gene expression, control cell growth and propagation, the mutation meeting of C-KIT genes Cause the generation of the malignant tumours such as acute myelocytic leukemia, gastrointestinal stromal tumor and carcinoma of testis.Clinical research shows intestines and stomach The effect of catastrophe of C-KIT genes is with Imatinib molecular targeted therapies in mesenchymoma is closely related.There are aobvious outside C-KIT Patient's curative effect of the mutation of son 11 is best, and patient's curative effect that extron 9 is mutated is taken second place, and GIST patient's curative effect without gene mutation is most Difference.American cancer integrated network（NCCN）《Soft tissue sarcoma's clinical therapeutic guideline》Middle proposition, GIST patient receive targeted drug Before treatment, C-KIT detection in Gene Mutation should be first carried out, decides whether to use targeted drug as clinical treatment according to testing result Measure.Detection C-KIT gene mutations have important ginseng for instructing rationally being treated using molecular targeted agents for GIST patient Examine value.

We recall the gene order before and after C-KIT gene D816V mutational sites from gene pool first, and will be mutated position Point is marked with double underline, in the upstream and downstream appropriate location in mutational site（Indicated with boldface type and underscore）, a pair of gram of design Grand primer, amplified fragments 116bp, mutational site are included in it, and gene order shows as follows, SEQ No.1：

tttgatttttatttttggtgtactgaatactttaaaacaaaagtattggattttttataatataagcaacactatag tattaaaaagttagttttcactctttacaagttaaaatgaatttaaatggttttcttttctcctccaacctaatagt gtattcacagagacttggcagccagaaatatcctccttactcatggtcggatcacaaagatttgtgattttggtcta gccagagacatcaagaatgattctaattatgtggttaaaggaaacgtgagtacccattctctgcttgacagtcctgc aaaggatttttagtttcaactttcgataaaaattgtttcctgtgattttcataatgtaaatcctgtctagggatatc acacattttagcagtcaaattaagtatacttcagcaaaatttgcatggtatgctgaacattactacaactaacattc aataatagaagtcctaattctaattgtgtaattttggggcatgtgaagg

Experimental procedure is as follows：

1）Extract genomic DNA

With the genome DNA extracting reagent kit of domestic Tiangeng company or Qiagen companies, genomic DNA, specific behaviour are extracted Make the progress of step by specification.The DNA samples prepared, through ultraviolet specrophotometer measured concentration, its concentration is adjusted to 50ng/ μ l, are stored in -20 DEG C, or directly carry out following reaction.

2）AT clones obtain positive plasmid

Cloning primer is designed in catastrophe point upstream and downstream first, is shown in Table 1：

1. C-KIT gene D816V mutational sites Wild type clone primer of table

In the PCR amplification system of 2 × PCR Master Mix (Fermentas) of 12.5 μ l, add 10 μM of upstreams and draw Thing and 10 μM of anti-sense primers, and genomic DNA 50ng, add water to 25 μ l, PCR reaction conditions of cumulative volume and are set as 95 DEG C of pre- changes Property 3min, 95 DEG C denaturation 10s, 60 DEG C annealing and extension 30s, altogether 35 circulation, take 15 μ l reaction products after reaction, into The gel electrophoresis of row concentration 2.5%, is observed after electrophoresis, and band is consistent with the size predicted, shows to expand successfully.Using U.S. The pCR2.0 TOPO carrier Ts of Life companies of state, by its operating instruction, by way of TA clones, obtain C-KIT genes D816V mutational sites wild-type positive plasmid, specific gene order is as follows, and it is correct that sequencing proof obtains plasmid.

SEQNo.4：ccttactcatggtcggatcacaaagatttgtgattttggtctagccagagacatcaagaat gattctaattatgtggttaaaggaaacgtgagtacccattctctgcttgacagtc

3）Rapid mutation method obtains mutant plasmid

Then, using the method for producing rapid mutation（Quickchange, QC）, mutant primer is designed, due to C-KIT D816V sites are the change of a to t, and specific primer sequence is shown in Table 2.

Table 2. obtains C-KIT gene D816V mutational sites mutant clones primer

According to（Stratagene companies）The reaction condition and step of Quickchange kits, complete the structure of mutant Build, obtained recombinant plasmid, specific gene order is as follows, and through sequence verification, it is saltant type, then by saltant type and open country Raw type plasmid is put -20 DEG C and is saved backup respectively.

SEQ No.7：ccttactcatggtcggatcacaaagatttgtgattttggtctagccagagtcatcaagaa tgattctaattatgtggttaaaggaaacgtgagtacccattctctgcttgacagtc

Embodiment 2：Allele-specific primers（ASP）Design and specificity screening

For C-KIT gene D816V mutational sites, design wild type and a series of mutation Idiotype primers are as follows：

C-KIT-D816V-WT-F: gatttgtgattttggtctagccagtga（SEQ No.8）

C-KIT-D816V-mut-F: gatttgtgattttggtctagccagtgt（SEQ No.9）

C-KIT-D816V-mut-F1: atttgtgattttggtctagccagtgt（SEQ No.10）

C-KIT-D816V-mut-F2: tttgtgattttggtctagccagtgt（SEQ No.11）

C-KIT-D816V-mut-F3: gatttgtgattttggtctagccagact（SEQ No.12）

C-KIT-D816V-mut-F4: atttgtgattttggtctagccagact（SEQ No.13）

C-KIT-D816V-mut-F5: tttgtgattttggtctagccagact（SEQ No.14）

Synthesis Taqman specific probe C-KIT- D816V-Probe are designed at the same time：

SEQ No.15：FAM-aaaggaaacgtgagtacccattctctgctt-BHQ1.

Relevant primer and probe are in raw work bioengineering（Shanghai）Co., Ltd is synthesized.

Then respectively with above-mentioned 7 primers and C-KIT-D816V-R: 5’- gactgtcaagcagagaatgggtac - 3’（SEQ No.16）Primer matches, and plus Taqman specific probes, adds the obtained wild type restructuring matter of foregoing structure About 30,000 copies of grain are template, and primer specificity screening is carried out on fluorescence quantitative PCR instrument, and reaction condition is set as the first step 95 DEG C of 2min pre-degenerations, 95 DEG C of 5s, 63 DEG C of 30s totally 15 circulations, do not collect fluorescence signal, second step 95 DEG C of 5s, 60 DEG C 30s totally 40 circulations, collect fluorescence signal, the result is shown in Figure 1.

Wherein, 1 ~ 7 curve represents C-KIT-D816V-WT-F, C-KIT-D816V-mut-F, C-KIT- respectively in Fig. 1 D816V-mut-F1、C-KIT-D816V-mut-F2、C-KIT-D816V-mut-F3、C-KIT-D816V-mut-F4、C-KIT- The PCR amplification curve of D816V-mut-F5, we have observed that No. 1 wild primers produce amplification letter in 19.5 circulations from Fig. 1 Number, 2-6 primers it is specific poor, respectively 20.5-24 circulate when produce non-specific amplification, same to wild primers（1 Number）Amplification efficiency difference is less than 19 circulations, and No. 7 primer meets our requirement, when circulating for 40, does not still expand Increase, 19 circulations are differed by more than with wild primers amplification efficiency.In order to have more preferable comparativity with mutant primer, our references The primer of No. 7 Primer redesign wild type is：tttgtgattttggtctagccagaca（SEQ No.17）.

Embodiment 3：ASP sensitivity is screened

Again with the mutant primers after screening respectively with the common anti-sense primer SEQ in C-KIT gene D816V mutational sites No.16 is matched, with saltant type recombinant plasmid, according to 10⁶, 10⁵, 10⁴, 10³, 10², 10,0 make gradient dilution, special plus Taqman Specific probes, in the enterprising line sensitivity verification of fluorescence quantitative PCR instrument.Mutant-specific primers can detect the prominent of 100 copies Variation, therefore this primer is exactly the optimal primer that detection C-KIT gene D816V mutational sites are filtered out by our method, It is shown in Table 3.

Embodiment 4：Prefabricated PCR of the present invention（Past-PCR）Reaction tube

According to the requirement of each two reaction tubes in mutational site, i.e. pipe detection wild type site, another pipe detection mutation Site, the primed probe of suitable concentration is coated in the milky PCR reaction tubes of 0.2ML in advance.

There is false negative when detecting in order to prevent, be coated with detection house-keeping gene GAPDH in advance in each reaction tube Sense primer, anti-sense primer and probe, wherein, GAPDH gene orders are as follows：

cctccgggaaactgtggcgtgatggccgcggggctctccagaacatcatccctgcctctactggcgctgccaaggct gtgggcaaggtcatccctgagctgaacgggaagctcactggcatggccttccgtgtccccactgccaacgtgtcagt ggtggacctgacctgccgtctagaaaaacctgccaaatatgatgacatcaagaaggtggtgaagcaggcgtcggagg gccccctcaagggcatcctgggctacactgagcaccaggtggtctcctctgacttcaacagcgacacccactcctcc acctttgacgctggggctggcattgccctcaacg（SEQ No.18）

The upstream primer sequence of detection house-keeping gene GAPDH is：ggctgtgggcaaggtcatc（SEQ No.19）

The downstream primer sequence of detection house-keeping gene GAPDH is:ctccgacgcctgcttcaccac(SEQ No.20）

The probe sequence of detection house-keeping gene GAPDH is：ccttccgtgtccccactgccaacgt(SEQ No.21)

Wherein, the end of probe 5 ' the HEX fluorescent markers of house-keeping gene GAPDH are detected, 3 ' ends are marked with BHQ, due to HEX with VIC belongs to same Air conduct measurement, and many instruments are often indicated using VIC, therefore is described in text with VIC.

Concrete details is shown in Table 3 in PCR reaction tubes.

The component and concentration of the prefabricated C-KIT genes D816V mutational sites Past-PCR reaction tubes of table 3.

Since we are in advance saltant type and wild type sense primer, probe and common anti-sense primer, detection house-keeping gene The upstream and downstream primer of GAPDH and the probe of detection house-keeping gene GAPDH are coated in the differential responses pipe for the number of finishing, and are effectively kept away Exempt from the operating error of user, while substantially increased operating efficiency, greatly facilitate actual use clinically.

Embodiment 5：The verification of sample

Gather melanoma patient tissue specimen, and use the genome DNA extracting reagent kit of Qiagen companies, extraction base Because of a group DNA, concrete operation step by specification carries out.The DNA samples prepared, will through ultraviolet specrophotometer measured concentration It is spare that its concentration is adjusted to 100ng/ μ l.

We use this mutant-specific primers filtered out, while are compared with wild primers, anti-in same PCR Answer on plate, each two reaction tube detects a gene loci, one of them is mutational site, the other is wild type site.Often 10mM Tris-HCl pH8.3,50mM KCl, 0.1%Triton X-100,2.0mM are included in a 20 μ l reaction volumes of reaction tube MgCl₂, the DNA of 100ng melanoma patients, 100nM specificity T aqman probes, 500nM shares specific downstream primer, and 500nM wild type specific forward primers, or saltant type specific forward primer, the upstream of 5pm detection house-keeping genes GAPDH are drawn Thing, the anti-sense primer of 5pm detection house-keeping genes GAPDH, the probe of 2.5pm detection house-keeping genes GAPDH, along with 1.5 units Taq archaeal dna polymerases, 200 μM of dNTPs, remaining is deionized water.

First step PCR reaction conditions are：37 DEG C of 2min, 95 DEG C of 2min pre-degenerations, then 95 DEG C of 5s, 63 DEG C of 30s, 15 Circulation, this step do not collect fluorescence；Second step PCR reaction conditions are：95 DEG C of 5s, 60 DEG C of 30s, totally 40 circulations, this step are collected glimmering Light.

1st, mutation allele homozygote is carried out according to the method described above（T sites）Detection, its specific gene order are：

SEQ No.22：ctagccagagtcatcaagaat

The FAM passage PCR amplification curve maps being collected into, as shown in Fig. 2, wherein, it is anti-that 1 curve represents detection saltant type PCR Amplification curve that should be in pipe, 2 curves represent the amplification curve in detection wild type PCR reaction tubes.

2nd, wild allele genic homozygote is carried out according to the method described above（A sites）Detection, its specific gene order are：

SEQ No.23：ctagccagagacatcaagaat

The FAM passage PCR amplification curve maps being collected into, as shown in figure 3, wherein, it is anti-that 3 curves represent detection wild type PCR Amplification curve that should be in pipe, 4 curves represent the amplification curve in detection saltant type PCR reaction tubes.

Meanwhile it is above-mentioned 2 detection case in, when VIC passage house-keeping genes GAPDH have logarithm increase amplification curve, such as Shown in Fig. 4, while FAM passages have the amplification curve that logarithm increases, then this time experimental result is effective.

Therefore, from above-mentioned detection experiment as it can be seen that the testing result of this kit is " all or none " as a result, even tested Sample is mutation allele homozygote, and in two PCR reaction tubes, only saltant type PCR reaction tubes are in FAM passages and VIC Passage has the amplification curve that logarithm increases, and wild type PCR reaction tubes have no the amplification curve of logarithm growth in FAM passages； If tested sample is wild allele genic homozygote, in two PCR reaction tubes, only wild type PCR reaction tubes are in FAM passages There is the amplification curve of logarithm growth with VIC passages, and saltant type PCR reaction tubes have no the amplification of logarithm growth in FAM passages Curve.The testing result interpretation of the kit is simple, and reducing the testing result of conventional kit needs to carry out a systems such as Δ Ct The process of row complicated calculations, reduces false determination ratio, while also effectively prevent false there are false negative in conventional kit testing result The generation of positive phenomenon.In addition kit provided by the invention can be used for the fluorescence quantitative PCR instrument of any model, detecting instrument letter Single, cost is low, and strong instrument is provided for scientific research and clinic C-KIT gene D816V site partings and gene mutation analysis.

Sequence table

<110>Shenyang You Jinuo bio tech ltd

<120>Detect primer, kit and its PCR method in C-KIT gene D816V mutational sites

<130> 2015

<160> 23

<170> PatentIn version 3.3

<210> 1

<211> 511

<212> DNA

<213>It is artificial synthesized

<400> 1

tttgattttt atttttggtg tactgaatac tttaaaacaa aagtattggattttttataa 60

tataagcaac actatagtat taaaaagtta gttttcactc tttacaagttaaaatgaatt 120

taaatggttt tcttttctcc tccaacctaa tagtgtattc acagagacttggcagccaga 180

aatatcctcc ttactcatgg tcggatcaca aagatttgtg attttggtctagccagagac 240

atcaagaatg attctaatta tgtggttaaa ggaaacgtga gtacccattctctgcttgac 300

agtcctgcaa aggattttta gtttcaactt tcgataaaaa ttgtttcctgtgattttcat 360

aatgtaaatc ctgtctaggg atatcacaca ttttagcagt caaattaagtatacttcagc 420

aaaatttgca tggtatgctg aacattacta caactaacat tcaataatagaagtcctaat 480

tctaattgtg taattttggg gcatgtgaag g 511

<210> 2

<211> 21

<212> DNA

<213>It is artificial synthesized

<400> 2

ccttactcat ggtcggatca c 21

<210> 3

<211> 24

<212> DNA

<213>It is artificial synthesized

<400> 3

gactgtcaag cagagaatgg gtac 24

<210> 4

<211> 116

<212> DNA

<213>It is artificial synthesized

<400> 4

ccttactcat ggtcggatca caaagatttg tgattttggt ctagccagagacatcaagaa 60

tgattctaat tatgtggtta aaggaaacgt gagtacccat tctctgcttgacagtc 116

<210> 5

<211> 28

<212> DNA

<213>It is artificial synthesized

<400> 5

gtctagccag agtcatcaag aatgattc 28

<210> 6

<211> 28

<212> DNA

<213>It is artificial synthesized

<400> 6

gaatcattct tgatgactct ggctagac 28

<210> 7

<211> 116

<212> DNA

<213>It is artificial synthesized

<400> 7

ccttactcat ggtcggatca caaagatttg tgattttggt ctagccagagtcatcaagaa 60

tgattctaat tatgtggtta aaggaaacgt gagtacccat tctctgcttg acagtc 116

<210> 8

<211> 27

<212> DNA

<213>It is artificial synthesized

<400> 8

gatttgtgat tttggtctag ccagtga 27

<210> 9

<211> 27

<212> DNA

<213>It is artificial synthesized

<400> 9

gatttgtgat tttggtctag ccagtgt 27

<210> 10

<211> 26

<212> DNA

<213>It is artificial synthesized

<400> 10

atttgtgatt ttggtctagc cagtgt 26

<210> 11

<211> 25

<212> DNA

<213>It is artificial synthesized

<400> 11

tttgtgattt tggtctagcc agtgt 25

<210> 12

<211> 27

<212> DNA

<213>It is artificial synthesized

<400> 12

gatttgtgat tttggtctag ccagact 27

<210> 13

<211> 26

<212> DNA

<213>It is artificial synthesized

<400> 13

atttgtgatt ttggtctagc cagact 26

<210> 14

<211> 25

<212> DNA

<213>It is artificial synthesized

<400> 14

tttgtgattt tggtctagcc agact 25

<210> 15

<211> 30

<212> DNA

<213>It is artificial synthesized

<400> 15

aaaggaaacg tgagtaccca ttctctgctt 30

<210> 16

<211> 24

<212> DNA

<213>It is artificial synthesized

<400> 16

gactgtcaag cagagaatgg gtac 24

<210> 17

<211> 25

<212> DNA

<213>It is artificial synthesized

<400> 17

tttgtgattt tggtctagcc agaca 25

<210> 18

<211> 342

<212> DNA

<213>It is artificial synthesized

<400> 18

cctccgggaa actgtggcgt gatggccgcg gggctctcca gaacatcatc cctgcctcta 60

ctggcgctgc caaggctgtg ggcaaggtca tccctgagct gaacgggaag ctcactggca 120

tggccttccg tgtccccact gccaacgtgt cagtggtgga cctgacctgccgtctagaaa 180

aacctgccaa atatgatgac atcaagaagg tggtgaagca ggcgtcggagggccccctca 240

agggcatcct gggctacact gagcaccagg tggtctcctc tgacttcaacagcgacaccc 300

actcctccac ctttgacgct ggggctggca ttgccctcaa cg 342

<210> 19

<211> 19

<212> DNA

<213>It is artificial synthesized

<400> 19

ggctgtgggc aaggtcatc 19

<210> 20

<211> 21

<212> DNA

<213>It is artificial synthesized

<400> 20

ctccgacgcc tgcttcacca c 21

<210> 21

<211> 25

<212> DNA

<213>It is artificial synthesized

<400> 21

ccttccgtgt ccccactgcc aacgt 25

<210> 22

<211> 21

<212> DNA

<213>It is artificial synthesized

<400> 22

ctagccagag tcatcaagaa t 21

<210> 23

<211> 21

<212> DNA

<213>It is artificial synthesized

<400> 23

ctagccagag acatcaagaa t 21

Claims

A kind of 1. reagent in detection C-KIT gene D816V mutational sites, it is characterised in that the reagent contain primer and with The probe that the primer is used cooperatively；

The primer includes：Wild type specific forward primer, saltant type specific forward primer and wild type specific upstream The anti-sense primer that primer is shared with saltant type specific forward primer；

And the wild type specific forward primer sequence is as shown in SEQ No.17, the saltant type specific forward primer sequence Row are as shown in SEQ No.14, and the shared downstream primer sequence is as shown in SEQ No.16；

The probe is Taqman probes, and sequence is as shown in SEQ No.15.
A kind of 2. kit in detection C-KIT gene D816V mutational sites, it is characterised in that：The kit is wanted containing having the right Seek the reagent described in 1.
3. according to the kit that C-KIT gene D816V mutational sites are detected described in claim 2, it is characterised in that：The reagent Box further includes PCR reaction tubes, dNTPs and Taq enzyme mixed liquor, PCR buffer solutions, the sense primer of detection house-keeping gene GAPDH, inspection Survey probe, positive quality control product and the blank control of the anti-sense primer, detection house-keeping gene GAPDH of house-keeping gene GAPDH；

The upstream primer sequence of the detection house-keeping gene GAPDH is as shown in SEQ No.19；The detection house-keeping gene GAPDH Downstream primer sequence as shown in SEQ No.20；The probe sequence of the detection house-keeping gene GAPDH is as shown in SEQ No.21.
4. according to the kit that C-KIT gene D816V mutational sites are detected described in claim 3, it is characterised in that：The mutation Type specificity sense primer, the shared anti-sense primer, the probe, the detection house-keeping gene GAPDH sense primer, The probe of the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene GAPDH are coated in T-shaped in advance In PCR reaction tubes；And with the wild type specific forward primer, the shared anti-sense primer, the probe, the inspection Survey the sense primer of house-keeping gene GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene The probe of GAPDH is coated in A type PCR reaction tubes in advance.
5. according to the kit that C-KIT gene D816V mutational sites are detected described in claim 4, it is characterised in that：It is described T-shaped Saltant type specific forward primer, the shared anti-sense primer, the probe, the detection house-keeping gene in PCR reaction tubes The probe of the sense primer of GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene GAPDH Concentration is respectively 50nM-2 μm, 50nM-2 μm, 50nM-400nM, 1pm-20pm, 1pm-20pm, 0.05pm-5pm；And the A Wild type specificity sense primer, the shared anti-sense primer, the probe, detection house keeper's base in type PCR reaction tubes Because of the spy of the sense primer of GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene GAPDH Pin concentration is respectively 50nM-2 μm, 50nM-2 μm, 50nM-400nM, 1pm-20pm, 1pm-20pm, 0.05pm-5pm.
6. according to the kit that C-KIT gene D816V mutational sites are detected described in claim 5, it is characterised in that：It is described T-shaped Saltant type specific forward primer, the shared anti-sense primer, the probe, the detection house-keeping gene in PCR reaction tubes The probe of the sense primer of GAPDH, the anti-sense primer of the detection house-keeping gene GAPDH and the detection house-keeping gene GAPDH Concentration is respectively 500nM, 500nM, 100nM, 5pm, 5pm, 2.5pm；And wild type specificity in the A types PCR reaction tubes Sense primer, the shared anti-sense primer, the probe, the sense primer of the detection house-keeping gene GAPDH, the detection The concentration and probe concentration of the anti-sense primer of house-keeping gene GAPDH and the detection house-keeping gene GAPDH be respectively 500nM, 500nM, 100nM、5pm、5pm、2.5pm。