WO2019084998A1 - 一种kras、nras、braf基因突变检测试剂盒 - Google Patents

一种kras、nras、braf基因突变检测试剂盒 Download PDF

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WO2019084998A1
WO2019084998A1 PCT/CN2017/110439 CN2017110439W WO2019084998A1 WO 2019084998 A1 WO2019084998 A1 WO 2019084998A1 CN 2017110439 W CN2017110439 W CN 2017110439W WO 2019084998 A1 WO2019084998 A1 WO 2019084998A1
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sequence
specifically
internal control
nras
kras
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French (fr)
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喻德华
翟建新
罗玄
李鑫剩
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深圳市优圣康生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the invention belongs to the technical field of medical detection, in particular to a DNA detection material, in particular to a KRAS, NRAS, BRAF gene mutation detection kit.
  • the products of the invention are useful for detecting gene mutations in KRAS/NRAS/BRAF of human tumors.
  • cancer is the result of interaction between environmental factors and cellular genetic material, is the result of multi-factor, multi-stage and multi-gene effects, and is the result of the accumulation of genetic mutations, so cancer is a genetic disease.
  • proto-oncogenes and tumor suppressor genes are closely related to the occurrence and development of cancer. Mutations in protooncogenes and/or tumor suppressor genes can cause cancerous cells.
  • the process of cancer-associated genetic mutations has occurred before cancer forms a solid mass, and the process may have been for more than a decade or more. During this period, the cancer cells have not formed a significant mass, but a small amount of tumor DNA has been released into the circulating blood.
  • Detection of mutations in these circulating genes by sequencing, PCR and other gene detection techniques can screen early or ultra-early cancers, evaluate the efficacy of radiotherapy and chemotherapy, predict the efficacy of targeted drugs, monitor early postoperative recurrence, etc.; Gene mutation detection helps to understand the tumor production and development process, determine the type and prognosis of the tumor, and help predict the efficacy of proto-oncogene-related anti-tumor drugs. Gene mutations often appear as heterozygous mutations, deletions or insertion mutations, and mutation detection requires high quality sequencing. By using the gene analyzer to perform mutation analysis, various mutation types such as heterozygous, deletion, and insertion mutation can be accurately determined, and the results are accurate and objective.
  • CRC Colorectal cancer
  • EGFR epidermal growth factor receptor
  • the KRAS gene mutation status is an effective indicator for predicting whether CRC patients can benefit from anti-EGFR therapy, but some KRAS wild-type gene carriers are still unable to benefit from anti-EGFR therapy, indicating RAS-RAF downstream of EGFR.
  • Other mutant genes present in the -MAPK and PI3-KAKT-mTOR signaling pathways eg, NRAS, BRAF, PIK3CA, and PTEN gene mutations may cause CRC patients to benefit from anti-EGFR therapy, so study KRAS/NRAS in CRC The mutation of the /BRAF gene will be of great significance for clinical treatment.
  • the tumor gene mutation detection technology used in clinical research mainly includes ARMS-PCR technology, first generation sequencing technology, second generation sequencing technology, digital PCR technology and the like.
  • the ARMS-PCR (AmplificationRefractory Mutation System) technique is a method for detecting a gene mutation by selectively amplifying a template using sequence-specific primers.
  • the core principle is that DNA polymerase-directed primer extension starts from the 3' end of the primer during PCR, and the degree of complementary pairing of the base at the 3' end of the primer with the template strongly influences the recognition of the polymerase and the progress of the PCR reaction: If the base is normally complementary to the template (AT, GC), the primer can be extended without interruption, and PCR can be performed efficiently to obtain a complete product; conversely, if the base is not properly paired with the template, the extension of the primer is blocked. Hysteresis, PCR efficiency is greatly reduced.
  • the conventional ARMS-PCR technology is easy to operate, the detection time is short, and the detection sensitivity is generally about 1%.
  • the sensitivity is low, the detection rate is significantly lower than the corresponding tumor tissue samples, and the flux is also detected. Not as good as sequencing methods.
  • Sequencing technology is the direct measurement of nucleic acid sequences to detect gene mutations. Sequencing technology has developed rapidly in recent years, from the first generation of sanger sequencing, to the second generation of high-volume sequencing, to the latest generation of single-molecule sequencing. The detection throughput and sensitivity have been greatly improved. Sanger sequencing method utilizes a certain proportion of dodepo-labeled dideoxynucleoside triphosphate (ddNTP) in the DNA synthesis reaction system to terminate the DNA synthesis and terminate at different nucleotides by denaturing polyacrylamide gel electrophoresis. The base sequence of the DNA to be tested is obtained by separation and development. The second-generation sequencing technology is also known as high-throughput sequencing technology, and the sequencing technologies of various companies have certain differences.
  • ddNTP dodepo-labeled dideoxynucleoside triphosphate
  • High-throughput sequencing can sequence hundreds of thousands to millions of DNA molecules in parallel, and the sequencing depth is much higher than that of the first-generation sequencing technology, but the fragment length is shorter.
  • the detection process of high-throughput sequencing is cumbersome, takes several days, and costs a lot.
  • Digital PCR is an absolute quantification technique for nucleic acid molecules.
  • the photometric method is based on the absorbance of nucleic acid molecules.
  • Real-time PCR is based on the Ct value.
  • the Ct value is the number of cycles in which the fluorescence value can be detected.
  • the digital PCR is the latest quantitative method.
  • Techniques, quantification of nucleic acids based on single-molecule PCR methods is an absolute quantitative method.
  • the microfluidic or microdroplet method of the current research field of analytical chemistry is mainly used to disperse a large amount of diluted nucleic acid solution into a microreactor or a droplet of a chip, and the number of nucleic acid templates per reactor is less than or equal to one.
  • the reactor with one template of the nucleic acid molecule gives a fluorescent signal, and the reactor without the template has no fluorescent signal.
  • the nucleic acid concentration of the original solution can be derived.
  • the method and reagents of the method are relatively high in cost, complicated in operation, and limited in flux, and are currently difficult to promote on the market.
  • the conventional ARMS-PCR technology is easy to operate, the detection time is short, and the detection sensitivity is generally about 1%.
  • the sensitivity is low, and the detection rate is significantly lower than the corresponding tumor tissue samples.
  • the amount is not as good as the sequencing method.
  • the detection specificity and flux of Sanger sequencing technology are high, but the sensitivity of the method itself is not high, and it is impossible to detect large fragment deletion rearrangement, and it can be sequenced by pre-PCR and electrophoresis, which is time-consuming and labor-intensive, and is not suitable for large-scale application.
  • the second-generation sequencing technology has greatly improved the sequencing depth, so the sensitivity is improved compared with the first-generation sequencing, but the detection cost, sensitivity and operation
  • the convenience of the work is not as good as the ARMS-PCR technology.
  • Digital PCR is applied to the detection of tumor gene mutations, and its technical principle determines its high detection sensitivity and specificity.
  • the main disadvantages are high cost, cumbersome operation, and low throughput.
  • One reaction can only detect one mutation site.
  • the invention aims at the shortcomings of the current detection method in terms of sensitivity, cost, flux, and ease of operation.
  • a highly sensitive design is developed by optimizing the reaction system and the amplification primers. High-throughput, low-cost, simple operation of colorectal cancer gene mutation detection reagents and kits. details as follows:
  • a KRAS, NRAS, BRAF gene mutation detection kit includes a detection reagent, and the detection reagent is composed of an internal control reagent, a mutation detection reagent and an external control reagent.
  • the internal control reagent includes an internal control primer R.
  • the sequence 5'-3' of the internal control primer R is specifically: GGCGCATTAATTACGGAAGAACCAGCT.
  • the mutation detecting reagents include: KRM1-R, KRM3-R, KRM4-R, KRex3-R, KRM5-R, NRex3-R, and NRM3-R; wherein
  • KRM1-R The sequence 5'-3' of KRM1-R is specifically: CGTCAAGGCACTCTTGCCTACGCCAT;
  • KRM3-R The sequence 5'-3' of KRM3-R is specifically: CGTCAAGGCACTCTTGCCTACGCCAA;
  • KRM4-R The sequence 5'-3' of KRM4-R is specifically: CGTCAAGGCACTCTTGCCTACGCCACT;
  • KRex3-R The sequence 5'-3' of KRex3-R is specifically: TTATGGCAAATACACAAAGAAA;
  • KRM5-R The sequence 5'-3' of KRM5-R is specifically: CGTCAAGGCACTCTTGCCTACGCCACG;
  • sequence 5'-3' of NRex3-R is specifically: CTCTCATGGCACTGTACTCTTCTTT;
  • NRM3-R The sequence 5'-3' of NRM3-R is specifically: AGCCCTTCGCCTGTCCTCATGTAT;
  • the external control reagent includes an externally controlled primer R.
  • sequence 5'-3' of the externally controlled primer R is specifically: CCAGGTTGGATCATATTCGTCCACAAA.
  • the internal control reagent of the detection reagent of the present invention further comprises: an internal control probe Pb, an internal control primer F and an external control primer F. among them,
  • the sequence 5'-3' of the internal control probe Pb is specifically: VIC-TCTTATACACATTGCTTTCT-MGB.
  • the sequence 5'-3' of the internal control primer F is specifically: GGCGCCTTCATGTTTATCCACTGTGTGGT;
  • sequence 5'-3' of the externally controlled primer F is specifically ATAGGCCCTGCTGAAAATGACTG;
  • Mutation detection reagents also include: KRM2-R, KRM6-R, KRM7-R, KRM9-R, BRex15-R, NRex2-R, KRM8-F, BRM1-F, NRM4-F, KRex2-F, KRex4-F, NRex3-F, NRM1-F and NRM2-F. among them,
  • KRM2-R The sequence 5'-3' of KRM2-R is specifically: CGTCAAGGCACTCTTGCCTACGCCAG;
  • KRM6-R The sequence 5'-3' of KRM6-R is specifically: CGTCAAGGCACTCTTGCCTACGCCACA;
  • the sequence 5'-3' of KRM7-R is specifically: TCGTCAAGGCACTCTTGCCTACGT;
  • KRM9-R The sequence 5'-3' of KRM9-R is specifically: AGTGTTACTTACCTGTCTTGTCTTTGT;
  • sequence 5'-3' of BRex15-R is specifically: TCCACAAAATGGATCCAGACAA;
  • sequence 5'-3' of NRex2-R is specifically: AAGGTGGTTCTGGATTAGCTGGAT;
  • the sequence 5'-3' of KRM8-F is specifically: GGATATTCTCGACACAGCAGGTCAC;
  • the sequence 5'-3' of BRM1-F is specifically: AAATAGGTGATTTTGGTCTAGCTACAGA;
  • NRM4-F The sequence 5'-3' of NRM4-F is specifically: CCGCATACTGGATACAGCTGGACG;
  • KRex2-F The sequence 5'-3' of KRex2-F is specifically: ATAGGGCCTGCTGAAAATGACTG;
  • KRex4-F The sequence 5'-3' of KRex4-F is specifically: GTGATTTGCCTTCTAGAACAGTAGAC;
  • sequence 5'-3' of NRex3-F is specifically: GGTGAAACCTGTTTGTTGGACATA;
  • NRM1-F The sequence 5'-3' of NRM1-F is specifically: CGCCCTGGTGGTGGTTGGAGCAT;
  • the sequence 5'-3' of NRM2-F is specifically: AACGGGTGGTGGTTGGAGCTGA.
  • the kit of the invention comprises an auxiliary reagent and a consumable.
  • auxiliary reagents include Taq enzyme and PCR reaction buffer.
  • the consumables are fluorescent PCR tubes.
  • the kit of the present invention is for detecting simultaneous detection of 14 mutation sites of three oncogenes KRAS, NRAS and BRAF associated with colorectal cancer.
  • the detection sensitivity can reach 0.1% (25ng DNA background), which is much higher than the traditional detection technology and is equivalent to digital PCR.
  • tumor tissue samples fresh tissue, FFPE, frozen tissue, etc.
  • body fluid samples such as blood, urine, etc.
  • test agent is composed of an internal control reagent, a mutation detection reagent and an external control reagent.
  • the internal control reagent includes an internal control primer R.
  • the sequence 5'-3' of the internal control primer R is specifically: GGCGCATTAATTACGGAAGAACCAGCT.
  • Mutation detection reagents include: KRM1-R, KRM3-R, KRM4-R, KRex3-R, KRM5-R, NRex3-R, and NRM3-R. among them,
  • KRM1-R The sequence 5'-3' of KRM1-R is specifically: CGTCAAGGCACTCTTGCCTACGCCAT;
  • KRM3-R The sequence 5'-3' of KRM3-R is specifically: CGTCAAGGCACTCTTGCCTACGCCAA;
  • KRM4-R The sequence 5'-3' of KRM4-R is specifically: CGTCAAGGCACTCTTGCCTACGCCACT;
  • KRex3-R The sequence 5'-3' of KRex3-R is specifically: TTATGGCAAATACACAAAGAAA;
  • KRM5-R The sequence 5'-3' of KRM5-R is specifically: CGTCAAGGCACTCTTGCCTACGCCACG;
  • sequence 5'-3' of NRex3-R is specifically: CTCTCATGGCACTGTACTCTTCTTT;
  • NRM3-R The sequence 5'-3' of NRM3-R is specifically: AGCCCTTCGCCTGTCCTCATGTAT;
  • the external control reagent includes: an external control primer R;
  • sequence 5'-3' of the externally controlled primer R is specifically: CCAGGTTGGATCATATTCGTCCACAAA.
  • the internal control reagent further includes an internal control probe Pb.
  • the sequence 5'-3' of the internal control probe Pb is specifically: VIC-TCTTATACACATTGCTTTCT-MGB.
  • the internal control reagent further includes an internal control primer F.
  • Mutation detection reagents also include KRM8-F and BRM1-F.
  • the external control reagent also includes an externally controlled primer F. among them,
  • the sequence 5'-3' of the internal control primer F is specifically: GGCGCCTTCATGTTTATCCACTGTGTGGT.
  • the sequence 5'-3' of KRM8-F is specifically: GGATATTCTCGACACAGCAGGTCAC;
  • the sequence 5'-3' of BRM1-F is specifically: AAATAGGTGATTTTGGTCTAGCTACAGA.
  • sequence 5'-3' of the externally controlled primer F is specifically ATAGGCCCTGCTGAAAATGACTG.
  • the mutation detecting reagent further includes: KRM2-R, KRM6-R, KRM7-R, KRM9-R, BRex15-R, and NRex2-R. among them,
  • KRM2-R The sequence 5'-3' of KRM2-R is specifically: CGTCAAGGCACTCTTGCCTACGCCAG;
  • KRM6-R The sequence 5'-3' of KRM6-R is specifically: CGTCAAGGCACTCTTGCCTACGCCACA;
  • the sequence 5'-3' of KRM7-R is specifically: TCGTCAAGGCACTCTTGCCTACGT;
  • KRM9-R The sequence 5'-3' of KRM9-R is specifically: AGTGTTACTTACCTGTCTTGTCTTTGT;
  • sequence 5'-3' of BRex15-R is specifically: TCCACAAAATGGATCCAGACAA;
  • sequence 5'-3' of NRex2-R is specifically: AAGGTGGTTCTGGATTAGCTGGAT.
  • the mutation detecting reagent further includes: NRM4-F, KRex2-F, KRex4-F, NRex3-F, NRM1-F, and NRM2-F. among them,
  • NRM4-F The sequence 5'-3' of NRM4-F is specifically: CCGCATACTGGATACAGCTGGACG;
  • KRex2-F The sequence 5'-3' of KRex2-F is specifically: ATAGGGCCTGCTGAAAATGACTG;
  • KRex4-F The sequence 5'-3' of KRex4-F is specifically: GTGATTTGCCTTCTAGAACAGTAGAC;
  • sequence 5'-3' of NRex3-F is specifically: GGTGAAACCTGTTTGTTGGACATA;
  • NRM1-F The sequence 5'-3' of NRM1-F is specifically: CGCCCTGGTGGTGGTTGGAGCAT;
  • the sequence 5'-3' of NRM2-F is specifically: AACGGGTGGTGGTTGGAGCTGA.
  • the internal control reagent of the present invention further comprises: an internal control probe Pb, an internal control primer F, and an external control primer F. among them,
  • the sequence 5'-3' of the internal control probe Pb is specifically: VIC-TCTTATACACATTGCTTTCT-MGB;
  • the sequence 5'-3' of the internal control primer F is specifically: GGCGCCTTCATGTTTATCCACTGTGTGGT;
  • sequence 5'-3' of the externally controlled primer F is specifically ATAGGCCCTGCTGAAAATGACTG.
  • Mutation detection reagents also include: KRM2-R, KRM6-R, KRM7-R, KRM9-R, BRex15-R, NRex2-R, KRM8-F, BRM1-F, NRM4-F, KRex2-F, KRex4-F, NRex3-F, NRM1-F and NRM2-F. among them,
  • KRM2-R The sequence 5'-3' of KRM2-R is specifically: CGTCAAGGCACTCTTGCCTACGCCAG;
  • KRM6-R The sequence 5'-3' of KRM6-R is specifically: CGTCAAGGCACTCTTGCCTACGCCACA;
  • the sequence 5'-3' of KRM7-R is specifically: TCGTCAAGGCACTCTTGCCTACGT;
  • KRM9-R The sequence 5'-3' of KRM9-R is specifically: AGTGTTACTTACCTGTCTTGTCTTTGT;
  • sequence 5'-3' of BRex15-R is specifically: TCCACAAAATGGATCCAGACAA;
  • sequence 5'-3' of NRex2-R is specifically: AAGGTGGTTCTGGATTAGCTGGAT;
  • the sequence 5'-3' of KRM8-F is specifically: GGATATTCTCGACACAGCAGGTCAC;
  • the sequence 5'-3' of BRM1-F is specifically: AAATAGGTGATTTTGGTCTAGCTACAGA;
  • NRM4-F The sequence 5'-3' of NRM4-F is specifically: CCGCATACTGGATACAGCTGGACG;
  • KRex2-F The sequence 5'-3' of KRex2-F is specifically: ATAGGGCCTGCTGAAAATGACTG;
  • KRex4-F The sequence 5'-3' of KRex4-F is specifically: GTGATTTGCCTTCTAGAACAGTAGAC;
  • sequence 5'-3' of NRex3-F is specifically: GGTGAAACCTGTTTGTTGGACATA;
  • NRM1-F The sequence 5'-3' of NRM1-F is specifically: CGCCCTGGTGGTGGTTGGAGCAT;
  • the sequence 5'-3' of NRM2-F is specifically: AACGGGTGGTGGTTGGAGCTGA.
  • the kit of the invention comprises an auxiliary reagent and a consumable.
  • the auxiliary reagent includes Taq enzyme and PCR reaction buffer;
  • the consumable is a fluorescent PCR tube.
  • the fluorescent PCR tube is of the 8-type, and each tube is numbered one by one to 8, wherein the internal control primer F, the internal control primer R and the internal control probe Pb are all added to the reaction tube 1 to 7; among them,
  • KRM1-R and KRM2-R are added to the reaction tube 1; KRex2-F is added to the reaction tubes 1 to 3, 5 and 8; KRM3-R, KRM6-R are added to the reaction tube 2; KRM4-R and KRM7- R is added to the reaction tube 3; KRex3-R, KRex4-F, KRM8-F and KRM9-R are added to the reaction tube 4; BRex15-R, KRM5-R and BRM1-F are added to the reaction tube 5; NRex3- F, NRM1-F and NRM3-R are added to the reaction tube 6; NRex2-R is added to the reaction tubes 6 and 7; NRex3-R, NRM2-F and NRM4-F are added to the reaction tube 7; the external control primer F It is added to the reaction tube 8; the external control primer R is added to the reaction tube 8.
  • the kit has a detection sensitivity of up to 0.1% in a 25 ng DNA background; can simultaneously detect 14 mutation sites of KRAS/NRAS/BRAF3 oncogenes; can be used for tumor tissue samples or body fluids Detection of the sample; the detection process after nucleic acid purification does not exceed 2 hours.
  • the kit of the present invention is used for the detection of a mutation site associated with colorectal cancer. Specifically, it is a detection of simultaneous detection of 14 mutation sites of three oncogenes KRAS, NRAS and BRAF associated with colorectal cancer.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • the paraffin-embedded tissue sample sections were extracted using the FFPE DNA extraction kit, and the extracted DNA was diluted to 2 ng/ul with TE.
  • the kit consists of a KBN8-linked PCR reaction strip and a KBN positive control.
  • each primer probe was sequentially added to an 8-tube tube to a final concentration of 100 to 400 nM.
  • the threshold value is set.
  • the Ct value of the SYBR channel is positive within 20, and the Ct value is between 20 and 22 is the gray area, and the Ct value is greater than 22 Negative.
  • the VIC channel is an internal control signal, and the SYBR and VIC have no amplification signal, which is invalid.
  • the No. 8 reaction tube is externally controlled, and the Ct value of the SYBR channel is between 10 and 16.
  • the template DNA is effective, otherwise the amount of the sample is increased or decreased.

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Abstract

针对现有试剂盒在灵敏度、成本、通量、操作方便性等方面存在不足的问题，提供一种KRAS、NRAS、BRAF基因突变检测试剂盒，包括检测试剂，检测试剂由内控试剂、突变检测试剂和外控试剂组成。

Description

一种KRAS、NRAS、BRAF基因突变检测试剂盒 技术领域
本发明属于医疗检测技术领域，尤其涉及DNA检测材料，具体为一种KRAS、NRAS、BRAF基因突变检测试剂盒。本发明的产物适用于检测人类肿瘤的KRAS/NRAS/BRAF的基因突变。
背景技术
研究表明，癌症是环境因素与细胞遗传物质相互作用的结果，是多因素、多阶段与多基因作用的结果，是基因突变累积的结果，因此癌症是基因病。在人类众多的基因中，原癌基因和抑癌基因与癌症的发生发展密切相关。原癌基因和/或抑癌基因的突变可引起细胞癌变。癌症在形成实性肿块以前，与癌症相关的基因突变的过程已经发生，而且这个过程可能已长达十余年、甚至更长时间。在这期间，癌细胞还没有形成明显的肿块，但已有微量肿瘤DNA释放到循环血液中。通过测序，PCR等基因检测技术检测循环血液中这些基因的突变，可以筛查早期或超早期癌症，评估放化疗的疗效，预测靶向药物的疗效，监测术后早期复发等；对肿瘤组织进行基因突变检测，有助于了解肿瘤的产生、发展过程，判断肿瘤的类型和预后；有助于预测原癌基因相关抗肿瘤药物的疗效。基因突变常表现为杂合突变、缺失或插入突变等，突变检测对测序的质量要求很高。通过应用基因分析仪进行突变分析，可准确判断出杂合、缺失、插入突变等各种突变类型，结果准确客观。
结直肠癌(colorectalcancer，CRC)是常见的消化道肿瘤之一，近年来应用基因检测的方法判断CRC预后及指导基因靶向治疗已成为重要手段。其中RAS基因是表皮生长因子受体(epidermalgrowthfactorreceptor，EGFR)信号通路中的关键性靶点，在CRC演变过程中发挥了重要作用。抗EGFR抗体(西妥昔单抗和帕尼单抗)主要与内源性配体竞争性结合EGFR，阻断下游的RAS信号通路产生抗肿瘤效应。研究显示只有携带RAS野生型的CRC患者才能从抗EGFR治疗中获益。因此KRAS基因突变状态成为预示CRC患者能否从抗EGFR治疗中获益的有效指标，但是仍然有一部分KRAS野生型基因携带者无法从抗EGFR治疗中获益，这表明在EGFR下游的RAS-RAF-MAPK和PI3-KAKT-mTOR信号传导通路中存在的其他突变基因(例如NRAS、BRAF、PIK3CA和PTEN基因突变)可能导致CRC患者无法从抗EGFR治疗中获益，因此，研究CRC中KRAS/NRAS/BRAF基因突变情况，将对临床治疗具有重要意义。可以明确中国人群KRAS、NRAS和BRAF基因的突变特征，指导临床CRC的个体化治疗，为选择合适的临床治疗方案提供依据。目前用于临床的肿瘤基因突变检测技术主要有ARMS-PCR技术，一代测序技术，二代测序技术，数字PCR技术等。
ARMS-PCR(AmplificationRefractoryMutationSystem)技术，是一种利用序列特异性引物对模板进行选择性扩增而达到检测基因突变的方法。其核心原理是根据PCR过程中DNA聚合酶引导的引物延伸从引物的3’末端开始，而引物3’末端的碱基与模板的互补配对程度强烈影响聚合酶的识别作用和PCR反应的进行：若这个碱基与模板正常互补配对(A-T，G-C)，则引物可以不间断延伸，PCR得以高效进行，得到完整的产物；反之，若这个碱基与模板非正常配对，则引物的延伸受到阻滞，PCR效率大大降低。常规的ARMS-PCR技术操作简便，检测时间较短，检测灵敏度一般在1％左右，在检测血液样本时，敏感性较低，检出率明显低于相应的肿瘤组织样本，同时检测通量也不如测序方法。
测序技术是对核酸的序列直接进行测定来检测基因突变的情况，测序技术近年来发展迅速，从第一代的sanger测序法，到第二代高量测序，再到最新一代的单分子测序，检测通量及灵敏度有了大幅提高。Sanger测序法利用在DNA合成反应体系中，混入一定比例带有同位素标记的双脱氧核苷三磷酸(ddNTP)，使DNA合成延伸终止在不同的核苷酸上，通过变性聚丙烯酰胺凝胶电泳分离和显影获得待测DNA的碱基序列。二代测序技术又称为高通量测序技术，各家公司的测序技术原理有一定差异。高通量测序一次能够并行对几十万到几百万条DNA分子进行序列测定，测序深度远高于一代测序技术，但测序的片段长度较短。但高通量测序的检测过程繁琐，耗时需要几天，成本也较高。
数字PCR是一种核酸分子绝对定量技术。当前核酸分子的定量有三种方法，光度法基于核酸分子的吸光度来定量；实时荧光定量PCR(RealTimePCR)基于Ct值，Ct值就是指可以检测到荧光值对应的循环数；数字PCR是最新的定量技术，基于单分子PCR方法来进行计数的核酸定量，是一种绝对定量的方法。主要采用当前分析化学热门研究领域的微流控或微滴化方法，将大量稀释后的核酸溶液分散至芯片的微反应器或微滴中，每个反应器的核酸模板数少于或者等于1个。这样经过PCR循环之后，有一个核酸分子模板的反应器就会给出荧光信号，没有模板的反应器就没有荧光信号。根据相对比例和反应器的体积，就可以推算出原始溶液的核酸浓度。该方法的仪器和试剂成本较高，操作繁琐，通量也有限，目前市场上很难推广。
此外，常规的ARMS-PCR技术操作简便，检测时间较短，检测灵敏度一般在1％左右，在检测血液样本时，敏感性较低，检出率明显低于相应的肿瘤组织样本，同时检测通量也不如测序方法。Sanger测序技术的检测特异性和通量均较高，但是方法本身灵敏度不高，也不能检测大片段缺失重排，且需预先PCR和电泳才可以测序，耗时耗力，不适合大规模的应用。二代测序技术在测序深度上有了很大的提高，因此敏感性比一代测序有了提升，但检测成本、灵敏度及操 作的方便性等方面均不如ARMS-PCR技术。数字PCR应用于肿瘤基因突变检测，其技术原理决定了其有着极高的检测灵敏度和特异性。主要劣势是成本高、操作繁琐，通量也较低，一个反应只能检测一个突变位点。
发明内容
本发明针对目前检测方法在灵敏度、成本、通量、操作方便性等方面存在的不足，在ARMS-PCR技术的基础上，通过对反应体系和扩增引物的优化，设计开发出一种高灵敏、高通量、低成本、操作简单的结直肠癌基因突变检测试剂、试剂盒。具体如下：
一种KRAS、NRAS、BRAF基因突变检测试剂盒，包括检测试剂，检测试剂由内控试剂、突变检测试剂和外控试剂组成。
内控试剂包括内控引物R。
内控引物R的序列5'-3'具体为：GGCGCATTAATTACGGAAGAACCAGCT。
突变检测试剂包括：KRM1-R、KRM3-R、KRM4-R、KRex3-R、KRM5-R、NRex3-R和NRM3-R；其中，
KRM1-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAT；
KRM3-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAA；
KRM4-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACT；
KRex3-R的序列5'-3'具体为：TTATGGCAAATACACAAAGAAA；
KRM5-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACG；
NRex3-R的序列5'-3'具体为：CTCTCATGGCACTGTACTCTTCTTT；
NRM3-R的序列5'-3'具体为：AGCCCTTCGCCTGTCCTCATGTAT；
外控试剂包括外控引物R。
外控引物R的序列5'-3'具体为：CCAGGTTGGATCATATTCGTCCACAAA。
进一步说，本发明所述的检测试剂的内控试剂还包括：内控探针Pb、内控引物F和外控引物F。其中，
内控探针Pb的序列5'-3'具体为：VIC-TCTTATACACATTGCTTTCT-MGB。
内控引物F的序列5'-3'具体为：GGCGCCTTCATGTTTATCCACTGTGTGGT；
外控引物F的序列5'-3'具体为ATAAGGCCTGCTGAAAATGACTG；
突变检测试剂还包括：KRM2-R、KRM6-R、KRM7-R、KRM9-R、BRex15-R、NRex2-R、KRM8-F、BRM1-F、NRM4-F、KRex2-F、KRex4-F、NRex3-F、NRM1-F和NRM2-F。其中，
KRM2-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAG；
KRM6-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACA；
KRM7-R的序列5'-3'具体为：TCGTCAAGGCACTCTTGCCTACGT；
KRM9-R的序列5'-3'具体为：AGTGTTACTTACCTGTCTTGTCTTTGT；
BRex15-R的序列5'-3'具体为：TCCACAAAATGGATCCAGACAA；
NRex2-R的序列5'-3'具体为：AAGTGGTTCTGGATTAGCTGGAT；
KRM8-F的序列5'-3'具体为：GGATATTCTCGACACAGCAGGTCAC；
BRM1-F的序列5'-3'具体为：AAATAGGTGATTTTGGTCTAGCTACAGA；
NRM4-F的序列5'-3'具体为：CCGCATACTGGATACAGCTGGACG；
KRex2-F的序列5'-3'具体为：ATAAGGCCTGCTGAAAATGACTG；
KRex4-F的序列5'-3'具体为：GTGATTTGCCTTCTAGAACAGTAGAC；
NRex3-F的序列5'-3'具体为：GGTGAAACCTGTTTGTTGGACATA；
NRM1-F的序列5'-3'具体为：CGCCCTGGTGGTGGTTGGAGCAT；
NRM2-F的序列5'-3'具体为：AACGGGTGGTGGTTGGAGCTGA。
本发明所述的试剂盒，包括辅助试剂和耗材。其中，辅助试剂包括Taq酶和PCR反应缓冲液。耗材为荧光PCR管。
本发明所述的试剂盒，用于对与结直肠癌相关的3个癌基因KRAS、NRAS和BRAF的14个突变位进行同时检测的检测。
为了更好地体现本发明的特点，现将各引物序列及反应瓶的配置，列表阐述如下：
Figure PCTCN2017110439-appb-000001
Figure PCTCN2017110439-appb-000002
本发明技术方案带来的有益效果
1.高灵敏度：检测灵敏度最高可达到0.1％(25ngDNA背景)，远高于传统检测技术，与数字PCR相当。
2.高通量：一次可检测KRAS/NRAS/BRAF3个癌基因的14个常见突变位点。
3.适用样本范围广：可用于肿瘤组织样本(新鲜组织，FFPE，冰冻组织等)，体液样本(如血液、尿液等)。
4.低成本：检测成本不及数字PCR的30％，性价比高。
5.操作方便：只需在反应管内加入DNA模板，即可上机完成检测，人工操作少，整个检测过程不超过2小时(不包括核酸纯化过程)。
具体实施方式
现对本发明的技术特点与优点进一步阐述如下：
一种KRAS、NRAS、BRAF基因突变检测试剂盒，包括用于基因突变的检测试剂，所述检 测试剂由内控试剂、突变检测试剂和外控试剂组成。
内控试剂包括内控引物R。
内控引物R的序列5'-3'具体为：GGCGCATTAATTACGGAAGAACCAGCT。
突变检测试剂包括：KRM1-R、KRM3-R、KRM4-R、KRex3-R、KRM5-R、NRex3-R和NRM3-R。其中，
KRM1-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAT；
KRM3-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAA；
KRM4-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACT；
KRex3-R的序列5'-3'具体为：TTATGGCAAATACACAAAGAAA；
KRM5-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACG；
NRex3-R的序列5'-3'具体为：CTCTCATGGCACTGTACTCTTCTTT；
NRM3-R的序列5'-3'具体为：AGCCCTTCGCCTGTCCTCATGTAT；
外控试剂包括：外控引物R；
外控引物R的序列5'-3'具体为：CCAGGTTGGATCATATTCGTCCACAAA。
进一步说，内控试剂还包括内控探针Pb。内控探针Pb的序列5'-3'具体为：VIC-TCTTATACACATTGCTTTCT-MGB。
进一步说，内控试剂还包括内控引物F。突变检测试剂还包括KRM8-F和BRM1-F。外控试剂还包括外控引物F。其中，
内控引物F的序列5'-3'具体为：GGCGCCTTCATGTTTATCCACTGTGTGGT。
KRM8-F的序列5'-3'具体为：GGATATTCTCGACACAGCAGGTCAC；
BRM1-F的序列5'-3'具体为：AAATAGGTGATTTTGGTCTAGCTACAGA。
外控引物F的序列5'-3'具体为ATAAGGCCTGCTGAAAATGACTG。
进一步说，突变检测试剂还包括：KRM2-R、KRM6-R、KRM7-R、KRM9-R、BRex15-R和NRex2-R。其中，
KRM2-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAG；
KRM6-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACA；
KRM7-R的序列5'-3'具体为：TCGTCAAGGCACTCTTGCCTACGT；
KRM9-R的序列5'-3'具体为：AGTGTTACTTACCTGTCTTGTCTTTGT；
BRex15-R的序列5'-3'具体为：TCCACAAAATGGATCCAGACAA；
NRex2-R的序列5'-3'具体为：AAGTGGTTCTGGATTAGCTGGAT。
进一步说，突变检测试剂还包括：NRM4-F、KRex2-F、KRex4-F、NRex3-F、NRM1-F和NRM2-F。其中，
NRM4-F的序列5'-3'具体为：CCGCATACTGGATACAGCTGGACG；
KRex2-F的序列5'-3'具体为：ATAAGGCCTGCTGAAAATGACTG；
KRex4-F的序列5'-3'具体为：GTGATTTGCCTTCTAGAACAGTAGAC；
NRex3-F的序列5'-3'具体为：GGTGAAACCTGTTTGTTGGACATA；
NRM1-F的序列5'-3'具体为：CGCCCTGGTGGTGGTTGGAGCAT；
NRM2-F的序列5'-3'具体为：AACGGGTGGTGGTTGGAGCTGA。
优选的技术方案是：本发明的内控试剂还包括：内控探针Pb、内控引物F和外控引物F。其中，
内控探针Pb的序列5'-3'具体为：VIC-TCTTATACACATTGCTTTCT-MGB；
内控引物F的序列5'-3'具体为：GGCGCCTTCATGTTTATCCACTGTGTGGT；
外控引物F的序列5'-3'具体为ATAAGGCCTGCTGAAAATGACTG。
突变检测试剂还包括：KRM2-R、KRM6-R、KRM7-R、KRM9-R、BRex15-R、NRex2-R、KRM8-F、BRM1-F、NRM4-F、KRex2-F、KRex4-F、NRex3-F、NRM1-F和NRM2-F。其中，
KRM2-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAG；
KRM6-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACA；
KRM7-R的序列5'-3'具体为：TCGTCAAGGCACTCTTGCCTACGT；
KRM9-R的序列5'-3'具体为：AGTGTTACTTACCTGTCTTGTCTTTGT；
BRex15-R的序列5'-3'具体为：TCCACAAAATGGATCCAGACAA；
NRex2-R的序列5'-3'具体为：AAGTGGTTCTGGATTAGCTGGAT；
KRM8-F的序列5'-3'具体为：GGATATTCTCGACACAGCAGGTCAC；
BRM1-F的序列5'-3'具体为：AAATAGGTGATTTTGGTCTAGCTACAGA；
NRM4-F的序列5'-3'具体为：CCGCATACTGGATACAGCTGGACG；
KRex2-F的序列5'-3'具体为：ATAAGGCCTGCTGAAAATGACTG；
KRex4-F的序列5'-3'具体为：GTGATTTGCCTTCTAGAACAGTAGAC；
NRex3-F的序列5'-3'具体为：GGTGAAACCTGTTTGTTGGACATA；
NRM1-F的序列5'-3'具体为：CGCCCTGGTGGTGGTTGGAGCAT；
NRM2-F的序列5'-3'具体为：AACGGGTGGTGGTTGGAGCTGA。
本发明所述的试剂盒，包括辅助试剂和耗材。其中，辅助试剂包括Taq酶和PCR反应缓冲液；耗材为荧光PCR管。
进一步说，在所述的试剂盒中，荧光PCR管为8联式，各管由在1逐个编号至8，其中，内控引物F、内控引物R和内控探针Pb均添加在反应管1～7中；其中，
KRM1-R和KRM2-R添加在反应管1中；KRex2-F添加在反应管1～3、5和8中；KRM3-R、KRM6-R添加在反应管2中；KRM4-R和KRM7-R添加在反应管3中；KRex3-R、KRex4-F、KRM8-F和KRM9-R添加在反应管4中；BRex15-R、KRM5-R和BRM1-F添加在反应管5中；NRex3-F、NRM1-F和NRM3-R添加在反应管6中；NRex2-R添加在反应管6和7中；NRex3-R、NRM2-F和NRM4-F添加在反应管7中；外控引物F添加在反应管8中；外控引物R添加在反应管8中。
进一步说，所述的试剂盒，在25ngDNA背景下，检测灵敏度最高可达到0.1％；能够同时对KRAS/NRAS/BRAF3个癌基因的14个突变位点进行检测；能够用于肿瘤组织样本或体液样本的检测；经过核酸纯化后的检测过程不超过2小时。
采用本发明所述的试剂盒用于对与结直肠癌相关的突变位点的检测。具体地说，是对与结直肠癌相关的3个癌基因KRAS、NRAS和BRAF的14个突变位进行同时检测的检测。
实施例一：
1)引物序列设计
引物名称 序列5'-3' 功能 反应管
TBPF GGCGCCTTCATGTTTATCCACTGTGTGGT 内控 1～7
TBPR GGCGCATTAATTACGGAAGAACCAGCT 内控 1～7
TBP-Pb VIC-TCTTATACACATTGCTTTCT-MGB 内控 1～7
KRM1-R CGTCAAGGCACTCTTGCCTACGCCAT 突变检测 1
KRM2-R CGTCAAGGCACTCTTGCCTACGCCAG 突变检测 1
KRex2-F ATAAGGCCTGCTGAAAATGACTG 突变检测 1～3,5,8
KRM3-R CGTCAAGGCACTCTTGCCTACGCCAA 突变检测 2
KRM6-R CGTCAAGGCACTCTTGCCTACGCCACA 突变检测 2
KRM4-R CGTCAAGGCACTCTTGCCTACGCCACT 突变检测 3
KRM7-R TCGTCAAGGCACTCTTGCCTACGT 突变检测 3
KRex3-R TTATGGCAAATACACAAAGAAA 突变检测 4
KRex4-F GTGATTTGCCTTCTAGAACAGTAGAC 突变检测 4
KRM8-F GGATATTCTCGACACAGCAGGTCAC 突变检测 4
KRM9-R AGTGTTACTTACCTGTCTTGTCTTTGT 突变检测 4
BRex15-R TCCACAAAATGGATCCAGACAA 突变检测 5
KRM5-R CGTCAAGGCACTCTTGCCTACGCCACG 突变检测 5
BRM1-F AAATAGGTGATTTTGGTCTAGCTACAGA 突变检测 5
NRex3-F GGTGAAACCTGTTTGTTGGACATA 突变检测 6
NRM1-F CGCCCTGGTGGTGGTTGGAGCAT 突变检测 6
NRM3-R CTCTCATGGCACTGTACTCTTCTTT 突变检测 6
NRex2-R AAGTGGTTCTGGATTAGCTGGAT 突变检测 6,7
NRex3-R AGCCCTTCGCCTGTCCTCATGTAT 突变检测 7
NRM2-F AACGGGTGGTGGTTGGAGCTGA 突变检测 7
NRM4-F CCGCATACTGGATACAGCTGGACG 突变检测 7
KRex2R CCAGGTTGGATCATATTCGTCCACAAA 外控 8
2)样本制备
使用FFPEDNA提取试剂盒提取石蜡包埋组织样本切片，将提取的DNA用TE稀释至2ng/ul。
3)仪器
ABI7500、nanodrop2000、高速离心机、低速离心机、恒温干浴器、漩涡震荡仪、冰箱。
4)试剂盒的组成
试剂盒由KBN8联PCR反应条、KBN阳性对照组成。
反应条每孔的基本组成为：1×PCRBuffer,2～8pmoldNTPs,1.5～4.0mMMgCl2,1×SybrGreenI，0.5～2UHSTaq,0.1～1UUNG酶。按表1)在8联管中依次加入各引物探针，终浓度为100～400nM。
5)检测过程
在8联条每个反应孔内加入5μl2ng/μl的待测样本，盖好8联条的管盖，按以下程序上机检测。
95℃，2～10min。
95℃，5～16sec，62℃，40～60sec；共计15个循环。
95℃，5～16sec，60℃，40～60sec，72℃，30～60sec(收集SYBR和VIC荧光信号)；共计25 个循环。
6)结果判定
阳性结果判定：根据荧光PCR扩增曲线的拐点设置阈值，1～7号反应管，SYBR通道Ct值在20以内为阳性结果，Ct值在20至22之间的为灰区，Ct值大于22的为阴性。
质量控制：VIC通道为内控信号，SYBR与VIC均无扩增信号的为无效结果。8号反应管为外控，SYBR通道Ct值在10至16之间的，模板DNA有效，否则需增加或减少上样量。

Claims (10)

  1. 一种KRAS、NRAS、BRAF基因突变检测试剂盒，包括检测试剂，其特征在于，检测试剂由内控试剂、突变检测试剂和外控试剂组成；
    内控试剂包括：内控引物R；
    内控引物R的序列5'-3'具体为：GGCGCATTAATTACGGAAGAACCAGCT；
    突变检测试剂包括：KRM1-R、KRM3-R、KRM4-R、KRex3-R、KRM5-R、NRex3-R和NRM3-R；其中，
    KRM1-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAT；
    KRM3-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAA；
    KRM4-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACT；
    KRex3-R的序列5'-3'具体为：TTATGGCAAATACACAAAGAAA；
    KRM5-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACG；
    NRex3-R的序列5'-3'具体为：CTCTCATGGCACTGTACTCTTCTTT；
    NRM3-R的序列5'-3'具体为：AGCCCTTCGCCTGTCCTCATGTAT；
    外控试剂包括：外控引物R；
    外控引物R的序列5'-3'具体为：CCAGGTTGGATCATATTCGTCCACAAA。
  2. 根据权利要求1所述的一种KRAS、NRAS、BRAF基因突变检测试剂盒，其特征在于，内控试剂还包括：内控探针Pb；内控探针Pb的序列5'-3'具体为：VIC-TCTTATACACATTGCTTTCT-MGB。
  3. 根据权利要求1所述的一种KRAS、NRAS、BRAF基因突变检测试剂盒，其特征在于，内控试剂还包括内控引物F；突变检测试剂还包括KRM8-F和BRM1-F；外控试剂还包括外控引物F；其中，
    内控引物F的序列5'-3'具体为：GGCGCCTTCATGTTTATCCACTGTGTGGT；
    KRM8-F的序列5'-3'具体为：GGATATTCTCGACACAGCAGGTCAC；
    BRM1-F的序列5'-3'具体为：AAATAGGTGATTTTGGTCTAGCTACAGA；
    外控引物F的序列5'-3'具体为ATAAGGCCTGCTGAAAATGACTG。
  4. 根据权利要求1所述的一种KRAS、NRAS、BRAF基因突变检测试剂盒，其特征在于，突变检测试剂还包括：KRM2-R、KRM6-R、KRM7-R、KRM9-R、BRex15-R和NRex2-R；其中，
    KRM2-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAG；
    KRM6-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACA；
    KRM7-R的序列5'-3'具体为：TCGTCAAGGCACTCTTGCCTACGT；
    KRM9-R的序列5'-3'具体为：AGTGTTACTTACCTGTCTTGTCTTTGT；
    BRex15-R的序列5'-3'具体为：TCCACAAAATGGATCCAGACAA；
    NRex2-R的序列5'-3'具体为：AAGTGGTTCTGGATTAGCTGGAT。
  5. 根据权利要求3所述的一种KRAS、NRAS、BRAF基因突变检测试剂盒，其特征在于，突变检测试剂还包括：NRM4-F、KRex2-F、KRex4-F、NRex3-F、NRM1-F和NRM2-F；其中，
    NRM4-F的序列5'-3'具体为：CCGCATACTGGATACAGCTGGACG；
    KRex2-F的序列5'-3'具体为：ATAAGGCCTGCTGAAAATGACTG；
    KRex4-F的序列5'-3'具体为：GTGATTTGCCTTCTAGAACAGTAGAC；
    NRex3-F的序列5'-3'具体为：GGTGAAACCTGTTTGTTGGACATA；
    NRM1-F的序列5'-3'具体为：CGCCCTGGTGGTGGTTGGAGCAT；
    NRM2-F的序列5'-3'具体为：AACGGGTGGTGGTTGGAGCTGA。
  6. 根据权利要求1所述的一种KRAS、NRAS、BRAF基因突变检测试剂盒，其特征在于，内控试剂还包括：内控探针Pb、内控引物F和外控引物F；其中，
    内控探针Pb的序列5'-3'具体为：VIC-TCTTATACACATTGCTTTCT-MGB；
    内控引物F的序列5'-3'具体为：GGCGCCTTCATGTTTATCCACTGTGTGGT；
    外控引物F的序列5'-3'具体为ATAAGGCCTGCTGAAAATGACTG；
    突变检测试剂还包括：KRM2-R、KRM6-R、KRM7-R、KRM9-R、BRex15-R、NRex2-R、KRM8-F、BRM1-F、NRM4-F、KRex2-F、KRex4-F、NRex3-F、NRM1-F和NRM2-F；其中，
    KRM2-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCAG；
    KRM6-R的序列5'-3'具体为：CGTCAAGGCACTCTTGCCTACGCCACA；
    KRM7-R的序列5'-3'具体为：TCGTCAAGGCACTCTTGCCTACGT；
    KRM9-R的序列5'-3'具体为：AGTGTTACTTACCTGTCTTGTCTTTGT；
    BRex15-R的序列5'-3'具体为：TCCACAAAATGGATCCAGACAA；
    NRex2-R的序列5'-3'具体为：AAGTGGTTCTGGATTAGCTGGAT；
    KRM8-F的序列5'-3'具体为：GGATATTCTCGACACAGCAGGTCAC；
    BRM1-F的序列5'-3'具体为：AAATAGGTGATTTTGGTCTAGCTACAGA；
    NRM4-F的序列5'-3'具体为：CCGCATACTGGATACAGCTGGACG；
    KRex2-F的序列5'-3'具体为：ATAAGGCCTGCTGAAAATGACTG；
    KRex4-F的序列5'-3'具体为：GTGATTTGCCTTCTAGAACAGTAGAC；
    NRex3-F的序列5'-3'具体为：GGTGAAACCTGTTTGTTGGACATA；
    NRM1-F的序列5'-3'具体为：CGCCCTGGTGGTGGTTGGAGCAT；
    NRM2-F的序列5'-3'具体为：AACGGGTGGTGGTTGGAGCTGA。
  7. 根据权利要求6所述的一种KRAS、NRAS、BRAF基因突变检测试剂盒，其特征在于，包括辅助试剂和耗材；其中，辅助试剂包括Taq酶和PCR反应缓冲液；耗材为荧光PCR管。
  8. 根据权利要求7所述的一种KRAS、NRAS、BRAF基因突变检测试剂盒，其特征在于，荧光PCR管为8联式，各管由在1逐个编号至8，其中，
    内控引物F、内控引物R和内控探针Pb均添加在反应管1～7中；
    KRM1-R和KRM2-R添加在反应管1中；
    KRex2-F添加在反应管1～3、5和8中；
    KRM3-R、KRM6-R添加在反应管2中；
    KRM4-R和KRM7-R添加在反应管3中；
    KRex3-R、KRex4-F、KRM8-F和KRM9-R添加在反应管4中；
    BRex15-R、KRM5-R和BRM1-F添加在反应管5中；
    NRex3-F、NRM1-F和NRM3-R添加在反应管6中；
    NRex2-R添加在反应管6和7中；
    NRex3-R、NRM2-F和NRM4-F添加在反应管7中；
    外控引物F和外控引物R添加在反应管8中。
  9. 根据权利要求7或8任一所述的一种KRAS、NRAS、BRAF基因突变检测试剂盒，其特征在于，在25ngDNA背景下，本试剂盒的检测灵敏度最高可达到0.1％；本试剂盒能够同时对KRAS/NRAS/BRAF3个癌基因的14个突变位点进行检测；本试剂盒能够用于肿瘤组织样本或体液样本的检测；本试剂盒经过核酸纯化后的检测过程不超过2小时。
  10. 采用权利要求1至6任一所述的KRAS/NRAS/BRAF基因突变检测试剂盒的用途，其特征在于，用于对与结直肠癌相关的3个癌基因KRAS、NRAS和BRAF的14个突变位进行同时检测。
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