基于荧光PCR法的RASSF1A基因甲基化定性检测方法开发  

Development of a Qualitative Detection Method for RASSF1A Gene Methylation Based on Fluorescence PCR

作　　者：林小洪 LIN Xiaohong(Xiamen Feishuo Biotechnology Co.,Ltd.,Xiamen 361100,China)

机构地区：[1]厦门飞朔生物技术有限公司,福建厦门361100

出　　处：《生物化工》2024年第5期155-158,共4页Biological Chemical Engineering

摘　　要：目的:使用高通量测序法辅助开发RAS相关区域家族1A(Ras-Association Domain Family 1A,RASSF1A)基因甲基化定性检测试剂盒(荧光PCR法)。方法:提取37例肺癌血浆样本DNA进行亚硫酸氢盐修饰转化,使用高通量测序方法检测样本的亚硫酸氢盐转化效率和RASSF1A基因CpG位点的甲基化水平,之后使用荧光PCR法检测样本RASSF1A基因的甲基化状态。结果:高通量测序显示样本DNA的平均转化效率为99.47%;根据CpG位点甲基化水平的双向聚类分析结果,筛选出第26~30号CpG位点作为荧光PCR法的检测位点,以甲基化水平5%作为阈值线,ΔCt Cut-off值为9,得出两种检测方法对37例样本的测定结果一致。结论:本研究借助高通量测序技术成功构建基于荧光PCR法的RASSF1A基因甲基化定性检测方法,操作简便、成本低、效率高,适用于临床RASSF1A基因甲基化状态常规化检测。Objective:This paper employs high-throughput sequencing to assist in the development of a qualitative detection kit for the methylation of ras-association domain family 1A(RASSF1A)gene(fluorescence PCR method).Methods:DNA from 37 lung cancer plasma samples is extracted and subjected to bisulfite modification.Highthroughput sequencing is used to detect the efficiency of bisulfite conversion and the methylation levels at CpG sites of the RASSF1A gene.The methylation status of the RASSF1A gene in the samples is also detected using the fluorescence PCR method.Results:The high-throughput sequencing shows that average conversion efficiency of sample DNA is 99.47%;based on the results of the two-way hierarchical clustering analysis of the methylation levels at CpG sites,CpG sites 26 to 30 are selected as the detection sites for fluorescence PCR method.With a methylation level threshold of 5%,and theΔCt Cut-off value of 9,the results of the two detection methods are consistent for the 37 samples.Conclusion:This study successfully establishs a qualitative detection method for RASSF1A gene methylation based on the fluorescence PCR method with the aid of high-throughput sequencing technology.The method is easy to operate,costeffective,and efficient,making it suitable for routine clinical detection of the methylation status of the RASSF1A gene.

关 键 词：高通量测序 RASSF1A 甲基化 荧光PCR法 

分 类 号：R446[医药卫生—诊断学]