BRAF基因V600E突变检测的荧光PCR方法的建立

目的: 建立检测鼠类肉瘤滤过性毒菌致癌同源体B1(BRAF)基因V600E突变的荧光聚合酶链式反应(PCR)方法,为黑色素瘤的药物靶向治疗提供新的基因检测方法。方法: 根据BRAF基因序列V600E位点区域设计特异性的引物和探针,建立该检测方法。检测BRAF基因V600E突变的黑色素瘤临床样本、BRAF基因V600E突变质粒和正常临床样本,评价方法的准确性、灵敏度和特异性。结果: BRAF基因V600E突变的黑色素瘤临床样本在V600E检测体系中具有特异性的扩增;检测灵敏度约为10¹拷贝/反应;正常临床样本在V600E检测体系中没有特异性的扩增,均是V600E突变阴性。结论: 建立的方法可以用于检测黑色素瘤临床样本中的BRAF基因V600E突变。

Objective: To establish a real-time fluorescence polymerase chain reaction(PCR)method for BRAF V600E mutation detection, so as to provide a new detection method for the target therapy of melanoma. Methods: According to BRAF V600E region, specific primers and probes were designed.The real-time fluorescence PCR method was established.The melanoma clinical samples, BRAF V600E mutant plasmids and normal clinical samples were tested for the evaluation of accuracy, sensitivity and specificity of the method. Results: The established method can accurately detect BRAF V600E mutation of the melanoma samples because of the specific amplification in the V600E detection system.The sensitivity can reach 10 copies per reaction.The results of normal clinical samples without the specific amplification in the V600E detection system are negative. Conclusion: The novel real-time fluorescence PCR method was proved to be useful for detecting BRAF V600E mutation in the clinical samples of melanoma.

[1] IKAWA S,FUKUI M,UEYAMA Y,et al.B-raf,a new member of the raf family,is activated by DNA rearrangement[J].Mol Cell Biol,1988,8(6):2651
[2] HALILOVIC E,SOLIT DB.Therapeutic strategies for inhibiting oncogenic BRAF signaling[J].Curr Opin Pharmacol,2008,8(4):419
[3] CHAPMAN PB,HAUSCHILD A,ROBERT C,et al.BRIM-3 study group:Improved survival with vemurafenib in melanoma with BRAF V600E mutation[J].N Engl J Med,2011,364(26):2507
[4] LEE JH,CHOI JW,KIM YS.Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma:a meta-analysis[J].Br J Dermatol,2011,164(4):776
[5] DAVIES H,BIGNELL GR,COX C,et al.Mutations of the BRAF gene in human cancer[J].Nature,2002,417(6892):949
[6] PAIK PK,ARCILA ME,FARA M,et al.Clinical characteristics of patients with lung adenocarcinomas harboring BRAF mutations[J].J Clin Oncol,2011,29(15):2046
[7] HAMFJORD J,STANGELAND AM,SKREDE ML,et al.Wobble enhanced ARMS method for detection of KRAS and BRAF mutations[J].Diagn Mol Pathol,2011,20(3):158
[8] MARCHANT J,MANGE A,LARRIEUX M,et al.Comparative evaluation of the new FDA approved THxIDTM-BRAF test with high resolution melting and sanger sequencing[J].BMC Cancer,2014,14(1):519
[9] IHLE MA,FASSUNKE J,KONIG K,et al.Comparison of high resolution melting analysis,pyrosequencing,next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations[J].BMC Cancer,2014,14:13
[10] PICHLER M,BALIC M,STADELMEYER E,et al.Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors[J].J Mol Diagn,2009,11(2):140
[11] DOTE H,TSUKUDA K,TOYOOKA S,et al.Mutation analysis of the BRAF codon 599 in malignant pleural mesothelioma by enriched PCR-RFLP[J].Oncol Rep,2004,11(2):361
[12] LILLEBERG SL,DUROCHER J,SANDERS C,et al.High sensitivity scanning of colorectal tumors and matched plasma DNA for mutations in APC,TP53,K-RAS,and BRAF genes with a novel DHPLC fluorescence detection platform[J].Ann N Y Acad Sci,2004,1022:250
[13] YANG H,HIGGINS B,KOLINSKY K,et al.RG7204(PLX4032),a selective BRAFV600E inhibitor,displays potent antitumor activity in preclinical melanoma models[J].Cancer Res,2010,70(13):5518