端粒酶逆转录酶启动子热点突变的ARMS-LNA-qPCR检测方法建立  

ARMS-LNA-qPCR method for detecting telomerase reverse transcriptase promoter hotspot mutation

作　　者：张洁 李方[2] 齐长海[2] 梁国威 ZHANG Jie;LI Fang;QI Chang-hai(Department of Clinical Laboratory,Peking University Aerospace Clinical College of Medicine,Beijing100049,China)

机构地区：[1]北京大学航天临床医学院检验科,北京100049 [2]航天中心医院病理科

出　　处：《中国实验诊断学》2020年第4期577-579,共3页Chinese Journal of Laboratory Diagnosis

摘　　要：目的建立一种定量检测端粒酶逆转录酶(TERT)启动子区-124bp(C/T)和-146bp(C/T)位点突变率的实时荧光定量PCR方法。方法以常规PCR扩增含有TERT热点突变位点的纯化产物作为检测标本,采用等位基因扩增阻滞原理(ARMS)的引物,并结合锁核酸(LNA)探针作为野生等位基因抑制子,建立实时荧光定量PCR的TERT启动子热点突变率检测方法(ARMS-LNA-qPCR)。在30例表观健康人确定检测下限。结果所建方法的突变率标准曲线为:-124bp(C/T)突变率%=10(0.262×ΔCt+4.325)(P<0.001,R2=0.999),-146bp(C/T)突变率%=10(0.261×△Ct+3.895)(P<0.001,R2=0.999)。在表观健康人中所确定的-124bp和-146bp检测下限分别是0.07%和0.05%。结论所建ARMS-LNA-qPCR的TERT热点突变检测方法具有定量检测突变率并适合临床常规开展的优势。采用普通PCR纯化产物作为检测标本,使得所建方法具有检测各种类型标本的普适性。Objective To develop a real-time PCR method for quantitative detection of telomerase reverse transcriptase(TERT)promoter region-124 bp(C/T)and-146 bp(C/T)mutation rates.Methods The purified products,the fragment carrying the TERT hotspot mutation amplified by general PCR,were used as test samples.Using Amplification refractory mutation system(ARMS)primer combined with the locked nucleic acid(LNA)probe as wild type allele inhibitor,a real-time PCR(ARMS-LNA-qPCR)for quantitative detection of TERT hotspot mutation rates was established.The lower limit of detection was determined in 30 apparently healthy subjects.Results The mutation rate standard curves of the established method were:-124 mutation rate%=10(0.262×ΔCt+4.325)(P<0.001,R2=0.999),-146 mutation rate%=10(0.261×ΔCt+3.895)(P<0.001,R2=0.999).The lower detection limits of-124 bp and-146 bp determined in apparently healthy individuals were 0.07%and 0.05%,respectively.Conclusion The ARMS-LNA-qPCR method for detecting TERT promoter hotspot mutation has the advantage of quantitative detection of mutation rate and suitable for clinical routine applications,as well as the universality for detecting various types of specimens given the PCR purified product is used as a test specimen.

关 键 词：端粒酶逆转录酶 -124bp(C/T) -146bp(C/T) 荧光定量PCR 肝细胞肝癌 

分 类 号：R446.7[医药卫生—诊断学]