CN102367478A - ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method - Google Patents
ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method Download PDFInfo
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Abstract
The invention relates to the field of molecular biology and aims to provide an ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and a detection method. The kit comprises a qPCR hybrid reaction solution, a locked nucleic acid retardant probe, a reference primer, an ARMS primer and a positive control sample, wherein the qPCR hybrid reaction solution comprises a PCR buffer solution, dNTPs (Deoxynucleotide Triphosphates), MgCl2, GoldStarbest Taq enzyme, a universal PCR reverse primer and a universal TaqMan probe. The kit provided by the invention can be used for rapidly and accurately detecting specific locus mutation of KRAS genes in various cancer tissues with high sensitivity, has high sensitivity, and can be used for detecting genome DNA with various tissue origins, specially free DNA segments adopting cell-free systems, such as blood serum and blood plasma, or other body fluid origins, wherein the genome DNA is derived from cell systems. Compared with direct sequencing and other mutation detection technologies, the kit and the detection method thereof have the advantages of strong specificity, high sensitivity, simplicity and rapidness in operation, high throughput, safety, definiteness and objectivity in result identification and the like for detecting the KRAS gene mutation.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of quick detection kit and detection method thereof that instructs the KRAS gene mutation typing of tumour individualized treatment.
Background technology
The RAS proto-oncogene is the transforming gene that the clone comes out from rat sarcoma virus at first; After in people finders' such as nineteen eighty-two Weinberg transitional cell bladder carcinoma cell line, activatory HRAS gene being arranged, caused the very big concern of people to RAS oncogene role in human tumor incidence and development process.The gene that the RAS gene family is relevant with human tumor has three kinds---and HRAS, KRAS and NRAS are positioned at respectively on 11,12 and No. 1 karyomit(e).Wherein, KRAS has the greatest impact to human cancer, and it is just as a kind of molecular switch: when the path that just often can control the regulating cell growth; Take place unusually during like the KRAS transgenation, the permanent activation of this gene makes signal conduction disturbance in the cell, causes cell to continue growth, thereby and stops apoptosis generation canceration.
The KRAS transgenation occurs in the early stage of tumour, and the KRAS gene height of primary tumor and MET is consistent.It is generally acknowledged that KRAS gene state can not change because of treatment.Sudden change has taken place in the KRAS gene in kinds of tumors, its incidence is about 40% in colorectal cancer, and carcinoma of the pancreas is that lung cancer is about 15% more than 90%.12 and 13 codons (>=90%) that occur in the 2nd exon are mainly concentrated in the KRAS transgenation, and it is active that these sudden changes have destroyed KRAS albumen intrinsic GTPase, thereby make KRAS albumen be in the continuous activity state.
The detection KRAS transgenation of rapid sensitive has great clinical meaning: the KRAS gene unconventionality occurs in (1) normal people's blood examination, prompting belongs to the tumour high risk population; (2) if the innocent tumour patient detects the KRAS transgenation, prompting has the possibility that cancerates; (3) a large amount of multicenter 3 clinical trial phases show that target new drug Erbitux (Erbitux, Cetuximab) and handkerchief Buddhist nun monoclonal antibody (Panitumumab) are only applicable to not have the KRAS gene wild-type patient of sudden change, and are invalid to KRAS sudden change patient." the state-run cancer integrated network of the U.S. (NCCN) colorectal cancer clinical practice guideline " (2008 the 3rd edition) spell out: the one, and all metastatic colorectal cancer patients all should detect KRAS gene state; The 2nd, have only KRAS wild-type patient just to advise accepting the treatment of EGFR suppressor factor such as Cetuximab (Cetuximab) and handkerchief Buddhist nun monoclonal antibody (panitumumab); Just say like U.S. Georgetown University Marshall: in the treatment of colorectal cancer, from then on colorectal cancer " is divided into two ": the KRAS gene is wild-type or mutant, makes traditional a kind of disease be divided into two kinds of independently diseases.KRAS mutant colorectal cancer patients accounts for 40%, and this type patient can not benefit from anti-EGFR targeted drug treatment, increases untoward reaction danger and medical expense on the contrary on foot; And all the other KRAS wild-type patients of about 60% just probably benefit from this type pharmacological agent." 09 year NCCN nonsmall-cell lung cancer clinical practice guideline " spells out: if sudden change has taken place when the KRAS gene, (Tarceva/ Tarceva/Erlotinib) carries out molecular targeted treatment then not advise patient using Te Luokai.Therefore, whether detection patient KRAS gene suddenlys change and becomes the prerequisite condition that can decision use anti-EGFR targeted drug.
Have high-caliber circulation dissociative DNA in the malignant neoplastic disease human peripheral, this DNA derives from malignant cell.Recently research has found from cancer patients's blood plasma or serum DNA that several kinds of TS gene alterations such as KRAS suddenly change, and shows that this is to detect the new way that tumor-related gene changes.Conveniently, the blood plasma Molecular Detection becomes a focus of tumor research rapidly owing to draw materials.But only there is the circulation dissociative DNA of trace in the tumour peripheral blood, and wherein the copy number of mutator gene is lower, so the blood plasma Molecular Detection is higher than organizing to the sensitivity requirement of detection technique.
The method that is used for the KRAS detection in Gene Mutation is a lot, comprises like dna direct order-checking, tetra-sodium order-checking (Pyrosequencing), sex change HPLC (HDPLC), high resolving power solubility curve technology (HRM), restriction small segment length polymorphism analysis method (RFLP) etc.Wherein the dna direct order-checking is the gold standard of sudden change detection.There is following shortcoming in this method: the susceptibility of detection is not high enough: " Lung Cancer " (" lung cancer ") that people such as Katsuhiko once published in August, 2005 goes up report; If the content of mutator gene accounts for 10% when following of genomic dna total amount, then detect existence less than the sudden change sample with direct sequencing; Time-consuming, complicated operating process requires height to operator; The operation of non-stopped pipe relates to the operation behind the pcr amplification, therefore is prone to contaminatedly, causes that the result's is undesirable; The interpretation subjectivity of sequencing result is strong; Once the sample size of experiment detection is limited, at most can only the 8-24 example.Therefore direct sequencing is difficult to generally carry out clinical.
Have high-caliber circulation dissociative DNA in the malignant neoplastic disease human peripheral, this DNA derives from malignant cell.Recently research has found from cancer patients's blood plasma or serum DNA that several kinds of TS gene alterations such as KRAS suddenly change, and shows that this is to detect the new way that tumor-related gene changes.Conveniently, the blood plasma Molecular Detection becomes a focus of tumor research rapidly owing to draw materials.But only there is the circulation dissociative DNA of trace in the tumour peripheral blood, and wherein the copy number of mutator gene is lower, so the blood plasma Molecular Detection is higher than organizing to the sensitivity requirement of detection technique.
Summary of the invention
The technical problem that the present invention will solve is, overcomes deficiency of the prior art, and a kind of KRAS gene mutation typing ARMS-qPCR detection kit is provided, with this solve in the existing sudden change detection technique, time-consuming, program is loaded down with trivial details and problem such as easy pollution.This test kit can be used for high-throughput, low-cost rapid detection KRAS sudden change; It is highly sensitive; High specificity; Applicable to clinical common sample such as fresh frozen tissue, paraffin organization, the high-sensitivity detection of trace sudden change in atraumatic serum or the plasma sample except that pathological tissue is provided especially.
Based on above-mentioned purpose, the present invention adopts following technical scheme:
A kind of ARMS-qPCR detection kit of the KRAS of being used for gene mutation typing is provided, and this test kit comprises qPCR mixed reaction solution, lock nucleic acid retardance probe, with reference to primer, ARMS primer and positive control sample; Wherein the qPCR mixed reaction solution comprises: PCR damping fluid, dNTPs, MgCl
2, Goldstarbest Taq enzyme, the general downstream primer of PCR and general TaqMan probe;
The final concentration of each component is respectively in the qPCR reaction system: the PCR damping fluid is 1 *; DNTPs is 0.01～1.5mM; MgCl
2Be 0.5～5mM; Goldstarbest Taq enzyme is 0.05U/ μ L; The general downstream primer of PCR is 0.1～1 μ M; General TaqMan probe is 0.1～1 μ M; Lock nucleic acid retardance probe is 0.9 μ M, be that 0.3 μ M, ARMS primer are that 0.3 μ M, positive control sample are 10～300ng/ μ l with reference to primer;
The sequence of the general downstream primer of said PCR is shown in SEQ ID NO A1;
The sequence of said general TaqMan probe is shown in SEQ ID NO A2;
Said lock nucleic acid retardance probe and wild template are mated fully, part base lock nucleination, and its terminal phosphateization, thus suppress KRAS transgenation district wild-type DNA cloning.Its sequence is shown in SEQ ID NO A0;
Said is the upstream primer of can increase simultaneously a KRAS wild-type and a mutated genes group DNA with reference to primer, and its sequence is shown in SEQ ID NO A3;
Said ARMS primer is any one in seven kinds of upstream primers, is used for the corresponding respectively KRAS of detection gene the 12nd codon GAT, GTT, GCT, TGT, AGT and these six kinds of mutants of CGT and No. 13 this a kind of mutation type of codon GAC; The sequence of these seven kinds of upstream primers is shown in SEQ ID NO A4, SEQ ID NO A5, SEQ ID NO A6, SEQ ID NO A7, SEQ ID NO A8, SEQ ID NO A9, SEQ ID NO A10; Article 7, the ARMS primer in the mutating alkali yl coupling of 3 ' terminal bases and mutant to be detected, increases one or two base mispairings in its 3 ' terminal 2-3 reciprocal position, to increase its specificity respectively simultaneously; Be used for six kinds of base substitution mutation (GGT → GAT of specific amplification KRAS gene the 12nd codon; GGT → GTT; GGT → GCT, 12GGT → TGT, the template DNA of GGT → AGT and GGT → CGT) and a kind of base substitution mutation of the 13rd codon (template DNA of codon 13GGC → GAC).
Said positive control sample is the plasmon DNA that has KRAS gene six kinds of mutation types of No. 12 codon and a kind of base substitution mutation type of 13 codons respectively.
Each primer sequence is listed below:
SEQ?ID?NO?A0：5’-TGGAGCTG
GTG
GCGTAGGC-PO4-3’
SEQ?ID?NO?A1：5’-ACCTCTATTGTTGGATCATATTCGTC-3’
SEQ?ID?NO?A2：5’-FAM-GAATTAGCTGTATCGTCAAGGCACTCT-BHQ1-3’
SEQ?ID?NO?A3：5’-CTGAATATAAACTTGTGGTAGTT-3’
SEQ?ID?NO?A4：5’-CTTGTGGTAGTTGGAGCTTA-3’
SEQ?ID?NO?A5：5’-AAACTTGTGGTAGTTGGAGCGGT-3’
SEQ?ID?NO?A6：5’-AACTTGTGGTAGTTGGAGCTGC-3’
SEQ?ID?NO?A7：5’-ATAAACTTGTGGTAGTTGGAGCTA-3’
SEQ?ID?NO?A8：5’-AACTTGTGGTAGTTGGAGCGT-3’
SEQ?ID?NO?A9：5’-ATAAACTTGTGGTAGTTGGAGCCC-3’
SEQ?ID?NO?A10：13Asp-3：5’-GTGGTAGTTGGAGCTGGTAA-3’
Further; The present invention also provides the KRAS gene mutation typing ARMS-qPCR detection method based on the aforementioned agents box; This method is a pair of with reference to primer, TaqMan probe and lock nucleic acid retardance probe to the KRAS gene design earlier; Design the ARMS primer respectively to the KRAS gene mutation site, add the qPCR mixed reaction solution in the reaction system, carry out the detection of quantitative fluorescent PCR with reference to primer or ARMS primer, lock nucleic acid retardance probe and template DNA to be checked.Specifically may further comprise the steps:
(1) design and the screening general TaqMan probe, the lock nucleic acid that contain No. the 12nd, KRAS gene and No. 13 codons block probe, with reference to primer and 7 kinds of ARMS primers;
(2) genomic dna that from acellular system or cell system, extracts is as template DNA;
(3) carry out the real-time fluorescence quantitative PCR amplification: the sudden change of each sample detects and divides 8 pipes to carry out; Add identical qPCR mixed reaction solution, lock nucleic acid retardance probe and template DNA in every pipe, every pipe adds a kind of with reference in primer or the 7 kinds of ARMS primers respectively, and the ARMS-qPCR that carries out the KRAS gene mutation typing detects;
(4) interpretation as a result:
Positive with reference to primer pipe amplification curve, various ARMS primer Guan Junwu amplification curves or itself and amplification curve Δ ct＞10 with reference to the primer pipe, this sample results is judged as wild-type;
Positive with reference to primer pipe amplification curve, corresponding ARMS primer pipe amplification curve is positive, and itself and with reference to amplification curve Δ ct≤7 of primer pipe, this sample results interpretation is a mutant;
Positive with reference to primer pipe amplification curve, corresponding ARMS primer pipe amplification curve is positive, and itself and with reference to amplification curve Δ ct＞7 of primer pipe, this sample results interpretation is suspicious mutant; All show that like duplicate detection amplification curve is positive for 3 times, this sample results interpretation is a mutant;
Negative with reference to primer pipe amplification curve, point out this sample extraction failure, need to extract again.
Template DNA is selected in the cast of fresh freezing or paraffin-embedded tumor tissues PBC, peripheral blood serum or blood plasma, body fluid, cavity, to extract in the said step (2).
The condition of carrying out pcr amplification reaction in the said step (3) is: 92～97 ℃ of preparatory sex change 10～15min; 92～97 ℃ of sex change 5～15s, 58～64 ℃ of annealing 40s, 40～50 circulations.
With respect to prior art, beneficial effect of the present invention is:
KRAS gene specific locus mutation in the various cancerous tissues of test kit quick, accurate, the high sensitive detection of ability of the present invention.Highly sensitive, can detect various tissue-derived genomic dnas, not only derive from cell system, especially adopt the dissociative DNA fragment in acellular system such as serum, blood plasma or other body fluid source.
Lock nucleic acid (locked nucleic acid; LNA) be a kind of novel few nucleic acid derivative; In the structure 2 one 0 of B-D one ribofuranose; The 4C position forms annular oxygen methylene bridge, sulphur methylene bridge or amine methylene bridge through the shrink effect, and the structure of furanose is locked in the N configuration of type in the C3, has formed inflexible condensation structure.LNA has advantage such as nontoxicity in the hybridization avidity powerful with DNA/RNA, antisense activity, nuclease-resistant ability, good water solubility and the body as a kind of new antisense nucleic acid.Thereby it can combine to prepare the LNA probe with DNA and RNA specifically, compares with dna probe, and the stability of its hybridization and specificity increase, and can improve the efficient and the sensitivity of gene diagnosis greatly.The present invention designs lock nucleic acid retardance probe; Its sequence and wild template are mated fully; Part base lock nucleination is used for the KRAS wild-type DNA that specificity combines template DNA, because of its terminal phosphateization; Thereby can't extend the non-specific amplification that suppresses the wild-type template, increase sensitivity and the specificity that sudden change detects greatly.
Amplification refractory mutation system (amplification refractory mutation system ARMS) was set up in 1989, was used for the known mutations gene is detected.This method is through design sudden change specificity ARMS primer, and the mutating alkali yl coupling of its 3 ' terminal bases and mutant to be detected is used for specific amplification sudden change template.The detection sensitivity of ARMS depends on specificity and the reaction conditions such as the enzyme of ARMS primer, the optimization of magnesium ion concentration etc.In order to increase the specificity of primer, reduce primer and extend with the mispairing of the wrong timing of target DNA, can be through introducing another one or two base mismatch in 2-3 base of primer 3 ' end, make it and template between the multiple mispairing of formation to stop the mistake extension.
The present invention will lock the wild retardance probe of nucleic acid (LNA) and ARMS sudden change Auele Specific Primer combines, and is used for the KRAS gene mutation typing.This detection kit designs to the detection of KRAS gene hot saltation zone, comprises totally 7 kinds of mutator gene types of KRAS gene the 12nd, 13 codons.This test kit comprises the 25ul qPCR reaction system through optimizing; The characteristic of this reaction system is all to contain the qPCR mixed reaction solution; TaqMan probe positive reference substance and the LNA retardance sequence that suppresses the amplification of wild-type KRAS genomic dna, just the ARMS primer is distinguished to special mutational site to some extent.
Probe used in the present invention is 5 ' the TaqMan probe of end FAM mark, the oligonucleotide of probe two ends difference mark fluorescent reporter group (R) and fluorescent quenching group (Q).When probe was complete, when promptly random state was hybridized state with no PCR product, the fluorescence that reporter group sends was absorbed by quenching group.In the ARMS-qPCR amplification procedure; 5 ' of Goldstarbest Taq enzyme end 5 prime excision enzyme activity is simultaneously also the probe cracking when special PCR product and TaqMan probe generation hybridization, and the photofluorometer that the fluorescence that reporter group discharged just can be built in the detection by quantitative appearance detects.PCR is every through a circulation, and fluorescent signal is also the same with the purpose fragment, and the process that has sync index to increase, the power of fluorescent signal have just been represented what of copy number of template DNA.Therefore the present invention not only can be used for simple qualitative detection, also can be used as the detection by quantitative of the concrete content of sample.
The advantage of comprehensive ARMS primer and LNA retardance probe, and the characteristics of real-time fluorescence quantitative PCR technology compare with sudden change detection technique such as direct order-checking, and the present invention is used to detect the KRAS transgenation and has following advantage:
1. high specificity: the ARMS primer of design is respectively to seven kinds of special mutant nucleotide sequences of KRAS gene the 12nd and the 13rd codon, the corresponding sudden change template DNA of ability specific amplification; 3 ' terminal 2-3 reciprocal position at the ARMS primer increases one or two base mispairings, can increase the specificity of ARMS primer; Technology of the present invention adds the lock nucleic acid retardance probe of wild-type KRAS gene specific in the PCR reaction system, thereby through suppressing the amplification of wild type gene group DNA cloning enrichment mutant DNA, has further improved the specificity that detects.
2. susceptibility is high: technology of the present invention adds wild-type KRAS gene lock nucleic acid retardance probe (LNA) in the PCR reaction solution, through suppressing the amplification of wild-type template specifically, thereby reaches the inrichment to trace sudden change template, the raising detection sensitivity; Present technique detects KRAS transgenation susceptibility and reaches 0.1-1% (being that the 0.1-1% that mutator gene group DNA reaches the total DNA of gene can detect); And direct sequencing detects the susceptibility of KRAS sudden change and is about 10% (be mutator gene group DNA reach 10% of the total DNA of gene just can detect).
3. testing process is the stopped pipe reaction, greatly reduces the possibility of pollution and result error.
4. simple to operate fast, inspect to from sample that obtain a result can be at 3 hours with interior completion by ready samples.And direct sequencing detects complex steps: censorship sample → extraction DNA → pcr amplification → checking PCR product (electrophoresis) → purified pcr product → directly order-checking; And the electrophoresis process of two PCR products of its experience; Opportunities for contamination is big, is not suitable for carrying out on a large scale in hospital.
5. interpretation as a result is clear and definite, objective; Also can carry out quantitative analysis if need to the result.
6. high-throughput once can detect 48 examples at most.
7. safety: do not comprise hazardous and noxious substances in the whole system, need not the aftertreatment of PCR product, operator and environment are not had harm.
Description of drawings
Fig. 1 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → GAT base substitution mutation of colorectal cancer positive sample with the ARMS-qPCR detection kit; It is negative that this sample confirms that through order-checking GGT → GAT suddenlys change; It is positive to confirm that through the T-A clone GGT → GAT suddenlys change, and the positive colony rate is about 3%;
Fig. 2 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → GTT base substitution mutation of colorectal cancer positive sample with the ARMS-qPCR detection kit, it is positive that this sample confirms that through order-checking GGT → GTT suddenlys change;
Fig. 3 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → GCT base substitution mutation of colorectal cancer positive sample with the ARMS-qPCR detection kit, it is positive that this sample confirms that through order-checking GGT → GCT suddenlys change;
Fig. 4 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → AGT base substitution mutation of intestinal cancer positive sample with the ARMS-qPCR detection kit; It is negative that this sample confirms that through order-checking GGT → AGT suddenlys change; It is positive to confirm that through the T-A clone GGT → AGT suddenlys change, and the positive colony rate is about 6%;
Fig. 5 is for detecting the amplification curve diagram of KRAS gene the 12nd codon GGT → TGT base substitution mutation of intestinal cancer positive sample with the ARMS-qPCR detection kit;
Fig. 6 is the amplification curve diagram of KRAS gene the 12nd codon GGT → CGT base substitution mutation for detecting positive control 12Arg with the ARMS-qPCR detection kit, and this mutant form is relatively more rare in Chinese knot rectum cancer crowd.
Fig. 7 is for detecting the amplification curve diagram of KRAS gene the 13rd codon GGT → GAT base substitution mutation of intestinal cancer positive sample with the ARMS-qPCR detection kit;
Fig. 8 is for detecting the amplification curve diagram of the KRAS gene wild-type of intestinal cancer positive sample with the ARMS-qPCR detection kit.
Embodiment
For making the present invention be more prone to understand, will further set forth practical implementation case of the present invention below.
Collect the wax stone tissue that clinical pathology is diagnosed as 100 routine patients of knot rectal adenocarcinoma (CRC), all do not accept Cetuximab (cetuximab) treatment before all patient's arts.Experimental applications under the extraction genomic dna is provided with from wax stone.Detect the common 7 kinds of base substitution mutations of KRAS gene the 12nd, 13 codon with ARMS-qPCR.
Design and screening can specific detection above-mentioned 7 kinds of base substitution mutations each one of specificity ARMS primer and with reference to one of primer; Design one of general downstream primer; Design is also screened one of general TaqMan probe; One of probe is blocked in design also screening LNA, and each probe, primer sequence are distinguished specific as follows:
The LNA sequence is shown in SEQ ID NO A0 in the sequence table: 5 '-TGGAGCTG
GTG
GCGTAGGC-PO4-3 '.
General downstream primer sequence is: SEQ ID NO A1:5 '-ACCTCTATTGTTGGATCATATTCGTC-3 '
The TaqMan probe sequence is: SEQ ID NO A2:5 '-FAM-GAATTAGCTGTATCGTCAAGGCACTCT-3 '
With reference to primer sequence be: SEQ ID NO A3:5 '-CTGAATATAAACTTGTGGTAGTT-3 '
To KRAS gene the 12nd codon GGT → GAT
The ARMS primer sequence of mutant is: SEQID NO A4:5 '-CTTGTGGTAGTTGGAGCTTA-3 '
To KRAS gene the 12nd codon GGT → GTT
The ARMS primer sequence of mutant is: SEQID NO A5:5 '-AAACTTGTGGTAGTTGGAGCGGT-3 '
To KRAS gene the 12nd codon GGT → GCT
The ARMS primer sequence of mutant is: SEQID NO A6:5 '-AACTTGTGGTAGTTGGAGCTGC-3 '
To KRAS gene the 12nd codon GGT → AGT
The ARMS primer sequence of mutant is: SEQID NO A7:5 '-ATAAACTTGTGGTAGTTGGAGCTA-3 '
To KRAS gene the 12nd codon GGT → TGT
The ARMS primer sequence of mutant is: SEQID NO A8:5 '-AACTTGTGGTAGTTGGAGCGT-3 '
To KRAS gene the 12nd codon GGT → CGT
The ARMS primer sequence of mutant is: SEQID NO A9:5 '-ATAAACTTGTGGTAGTTGGAGCCC-3 '
To KRAS gene the 13rd codon GGC → GAC
The ARMS primer sequence of mutant is: SEQID NO A10:13Asp-3:5 '-GTGGTAGTTGGAGCTGGTAA-3 '
1. the optimization of reaction system:
(1) under the optimization of the primer concentration situation that other condition is identical in reaction system, primer concentration being done the multiple proportions serial dilution from 0.1 μ mol/L to 1.6 μ mol/L respectively, is 0.3 μ mol/L through the analysis of test-results is relatively decided best primer concentration.
(2) under the optimization of the concentration and probe concentration situation that other condition is identical in reaction system; With concentration and probe concentration respectively from; True 0.05 μ mol/L to 0.5 μ mol/L does the multiple proportions serial dilution, compares through the analysis to test-results, confirms that best concentration and probe concentration is 0.1 μ mol/L.
(3) under the optimization of the annealing temperature situation that other condition is identical in reaction system, carry out grads PCR (56 ° to 64 ° annealing temperatures), compare, confirm that optimum annealing temperature is 60 ° through analysis to test-results.
(4) optimization of enzyme adds various enzymes in reaction system, compares through the analysis to test-results, confirms that best enzyme is a gold star TAQ enzyme.
Through optimizing reaction system is 25 μ l, comprises qPCR mix 20 μ l, and with reference to primer or each 0.75 μ l (final concentration 0.3 μ M) of ARMS primer, LNA blocks probe 2.25 μ l (final concentration 0.9 μ M) template DNA 2.0 μ l (final concentration 10-300ng/ μ l).Said qPCR mix component and final concentration thereof are:
PCR damping fluid final concentration is 1 *;
The dNTPs final concentration is 0.2mM;
General downstream primer final concentration is 0.3 μ M;
TaqMan probe final concentration is 0.1 μ M;
Goldstarbest Taq enzyme final concentration is 0.05U/ μ L;
MgCl
2Final concentration is 3.5mM;
ARMS-qPCR carries out on the ABI7500 detector.Each sample carries out 8 tube reactions, and every tube reaction adds common qPCR mix, LNA retardance probe and template DNA, and different has only with reference to primer or ARMS primer.The PCR reaction conditions is: 95 ℃ of preparatory sex change 10 minutes, 95 ℃ 15 seconds, 60 ℃ 40 seconds, amplified reaction 40 circulations are collected fluorescence 60 ℃ of 40 second stages.
The result is: in 100 routine samples, detect 20 routine samples altogether KRAS gene the 12nd codon GGT → GAT sudden change takes place; KRAS gene the 12nd codon GGT → GTT sudden change takes place in 4 routine samples; KRAS gene the 12nd codon GGT → GCT sudden change takes place in 2 routine samples; KRAS gene the 12nd codon GGT → AGT sudden change takes place in 3 routine samples, and KRAS gene the 13rd codon GGC → GAC sudden change takes place 11 routine samples, and KRAS gene the 12nd codon GGT → TGT sudden change takes place 2 routine samples.Wherein 3 routine samples detect interpretation through the ARMS-qPCR of KRAS gene mutation typing to be that the 12nd codon GGT → GAT suddenly change positive, and it is negative to be through sequencing result that GGT → GAT suddenly change, and through T-A clone confirmation GGT → GAT positive of suddenling change, the positive colony rate is about 3-7%; Wherein to detect interpretation be the 12nd codon GGT → positive this sample of AGT sudden change to 1 routine sample KRAS gene type ARMS-qPCR that to be through sequencing result that GGT → AGT suddenly change negative, confirms GGT → AGT positive of suddenling change through the T-A clone, and the positive colony rate is about 6%.All the other results all are consistent with direct sequencing result.
It is highly sensitive that The above results shows that ARMS-qPCR method of the present invention detects tumor tissues KRAS transgenation, can detect the trace sudden change template (＜10%) in the clinical sample; Its reliable results detects " gold standard " with sudden change: coincidence rate as a result >=95% that directly checks order.Said in addition method is quick, simple to operate, and interpretation as a result is objective, closes the border the reaction pollution and lacks, and is very suitable in clinical, carrying out on a large scale.
Claims (4)
1. be used for the ARMS-qPCR detection kit of KRAS gene mutation typing, it is characterized in that, this test kit comprises qPCR mixed reaction solution, lock nucleic acid retardance probe, with reference to primer, ARMS primer and positive control sample; Wherein the qPCR mixed reaction solution comprises: PCR damping fluid, dNTPs, MgCl
2, Goldstarbest Taq enzyme, the general downstream primer of PCR and general TaqMan probe;
The qPCR reaction system is 25ul, and the final concentration of each component is respectively: the PCR damping fluid is 1 *; DNTPs is 0.01～1.5mM; MgCl
2Be 0.5～5mM; Goldstarbest Taq enzyme is 0.05U/ μ L; The general downstream primer of PCR is 0.1～1 μ M; General TaqMan probe is 0.1～1 μ M; Lock nucleic acid retardance probe is 0.9 μ M, be that 0.3 μ M, ARMS primer are that 0.3 μ M, positive control sample are 10～300ng/ μ l with reference to primer;
The sequence of said lock nucleic acid retardance probe is shown in SEQ ID NO A0;
The sequence of the general downstream primer of said PCR is shown in SEQ ID NO A1;
The sequence of said general TaqMan probe is shown in SEQ ID NO A2;
Said is the upstream primer of can increase simultaneously a KRAS wild-type and a mutated genes group DNA with reference to primer, and its sequence is shown in SEQ ID NO A3;
Said ARMS primer is any one in seven kinds of upstream primers, is used for the corresponding respectively KRAS of detection gene the 12nd codon GAT, GTT, GCT, TGT, AGT and these six kinds of mutants of CGT and No. 13 this a kind of mutation type of codon GAC; The sequence of these seven kinds of upstream primers is shown in SEQ ID NO A4, SEQ ID NO A5, SEQ ID NO A6, SEQ ID NO A7, SEQ ID NO A8, SEQ ID NO A9, SEQ ID NO A10;
Said positive control sample is the corresponding respectively plasmon DNA that has aforementioned KRAS genic mutation type.
2. based on the KRAS gene mutation typing ARMS-qPCR detection method of the said test kit of claim 1, it is characterized in that, may further comprise the steps:
(1) design and the screening general TaqMan probe, the lock nucleic acid that contain No. the 12nd, KRAS gene and No. 13 codons block probe, with reference to primer and 7 kinds of ARMS primers;
(2) genomic dna that from acellular system or cell system, extracts is as template DNA;
(3) carry out the real-time fluorescence quantitative PCR amplification: the sudden change of each sample detects and divides 8 pipes to carry out; Add identical qPCR mixed reaction solution, lock nucleic acid retardance probe and template DNA in every pipe, every pipe adds a kind of with reference in primer or the 7 kinds of ARMS primers respectively, and the ARMS-qPCR that carries out the KRAS gene mutation typing detects;
(4) interpretation as a result:
Positive with reference to primer pipe amplification curve, various ARMS primer Guan Junwu amplification curves or itself and amplification curve Δ ct＞10 with reference to the primer pipe, this sample results is judged as wild-type;
Positive with reference to primer pipe amplification curve, corresponding ARMS primer pipe amplification curve is positive, and itself and with reference to amplification curve Δ ct≤7 of primer pipe, this sample results interpretation is a mutant;
Positive with reference to primer pipe amplification curve, corresponding ARMS primer pipe amplification curve is positive, and itself and with reference to amplification curve Δ ct＞7 of primer pipe, this sample results interpretation is suspicious mutant; All show that like duplicate detection amplification curve is positive for 3 times, this sample results interpretation is a mutant;
Negative with reference to primer pipe amplification curve, point out this sample extraction failure, need to extract again.
3. method according to claim 2 is characterized in that, template DNA is selected in the cast of fresh freezing or paraffin-embedded tumor tissues, PBC, peripheral blood serum or blood plasma, body fluid, cavity, to extract in the said step (2).
4. method according to claim 2 is characterized in that, the condition of carrying out pcr amplification reaction in the said step (3) is: 92～97 ℃ of preparatory sex change 10～15min; 92～97 ℃ of sex change 5～15s, 58～64 ℃ of annealing 40s, 40～45 circulations.
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