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一种基于RFLP-ARMS TaqMan PCR的新型方法用于检测黑色素瘤中的BRAF V600E突变。

A novel RFLP-ARMS TaqMan PCR-based method for detecting the BRAF V600E mutation in melanoma.

作者信息

Zhang Yunqing, Qu Shoufang, Zhao Jinyin, Yu Ting, Guo Liping, Yin Songchao, Hu Xiaoxu, Chen Weijun, Lai Wei, Huang Jie

机构信息

Department of Dermatology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, P.R. China.

Division of In Vitro Diagnostic Reagents, National Institutes for Food and Drug Control, Beijing 100050, P.R. China.

出版信息

Oncol Lett. 2018 Aug;16(2):1615-1621. doi: 10.3892/ol.2018.8844. Epub 2018 May 30.

DOI:10.3892/ol.2018.8844
PMID:30008844
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6036452/
Abstract

To enable the rapid and sensitive screening of the BRAF V600E mutation in clinical samples, a novel method combining restriction fragment length polymorphism (RFLP) analysis with the popular amplification refractory mutation system (ARMS) TaqMan quantitative (qPCR) genotyping method in a single reaction tube was developed. A total of 2 primer pairs were designed to enrich for and genotype the BRAF mutational hotspot (RFLP primers and ARMS primers) and a restriction enzyme was used to remove the wild-type alleles. The analysis revealed that this method detected mutant alleles in mixed samples containing >0.1% mutant sequences. In a survey of 53 melanoma samples, this method detected 21 mutation-positive samples. This novel RFLP-ARMS TaqMan qPCR protocol may prove useful for detecting mutations in clinical samples containing only a small proportion of mutant alleles.

摘要

为了能够在临床样本中快速、灵敏地筛查BRAF V600E突变，开发了一种新方法，该方法将限制性片段长度多态性（RFLP）分析与流行的扩增阻滞突变系统（ARMS）TaqMan定量（qPCR）基因分型方法结合在单个反应管中。总共设计了2对引物来富集BRAF突变热点并进行基因分型（RFLP引物和ARMS引物），并使用一种限制性酶去除野生型等位基因。分析表明，该方法可在含有>0.1%突变序列的混合样本中检测到突变等位基因。在对53个黑色素瘤样本的检测中，该方法检测到21个突变阳性样本。这种新的RFLP-ARMS TaqMan qPCR方案可能对检测仅含有少量突变等位基因的临床样本中的突变有用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aa6f/6036452/dc54c2181c58/ol-16-02-1615-g02.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aa6f/6036452/f76ed05b17a3/ol-16-02-1615-g00.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aa6f/6036452/c33e0be7f3b9/ol-16-02-1615-g01.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aa6f/6036452/dc54c2181c58/ol-16-02-1615-g02.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aa6f/6036452/f76ed05b17a3/ol-16-02-1615-g00.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aa6f/6036452/c33e0be7f3b9/ol-16-02-1615-g01.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/aa6f/6036452/dc54c2181c58/ol-16-02-1615-g02.jpg

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