基于突变扩增系统-实时荧光定量聚合酶链式反应法 高灵敏检测甲状腺癌中PTEN^(R130*)突变  

Highly Sensitive Detection of PTEN ^(R130*) Mutation in Thyroid Cancer Based on Amplification Refractory Mutation System-Real Time Fluorescence-Quantitative Polymerase Chain Reaction

作　　者：魏佳[1,2] 王瑶琪 王振新 孟宪瑛[1] WEI Jia;WANG Yao-Qi;WANG Zhen-Xin;MENG Xian-Ying(Department of Thyroid Surgery,The First Hospital of Jilin University,Changchun 130021,China;State Key Laboratory of Electroanalytical Chemistry,Changchun Institute of Applied Chemistry,Chinese Academy of Sciences,Changchun 130022,China)

机构地区：[1]吉林大学白求恩第一临床医院甲状腺外科,长春130021 [2]中国科学院长春应用化学研究所,电分析化学国家重点实验室,长春130022

出　　处：《分析化学》2022年第5期701-710,共10页Chinese Journal of Analytical Chemistry

基　　金：吉林省科技发展计划项目(No.20210204049YY);山东省泰山产业领军人才工程项目(No.2019TSCYCX-24)资助。

摘　　要：构建了一种基于突变扩增系统-实时荧光定量聚合酶链式反应(Amplification refractory mutation system-realtime fluorescence-quantitative polymerase chain reaction,ARMS-qPCR)的分析方法用于检测甲状腺癌中磷酸酶和张力蛋白同源性缺失(Phosphate and tension homology deleted on chromosome ten,PTEN)基因的PTEN ^(R130*)突变。针对PTEN ^(R130*)突变设计的等位基因特异性和非特异性引物,在两个不同的反应体系中对一份模板同时进行等位基因特异性和非特异性扩增,获得二者Ct值相减得到的ΔCt以实现对等位基因突变频率(Variant allele frequency,VAF)的分析。本研究所构建的ARMS-qPCR法检测线性范围为0.1%~90%VAF,能够检测低至0.01%的VAF,并识别基因组DNA中低至4拷贝的PTEN ^(R130*)突变。使用本方法在24例甲状腺肿瘤样品中成功检出5例VAF>0.1%的PTEN ^(R130*)突变。此外,ARMS-qPCR法对电化学富集后的PTEN ^(R130*)突变血浆加标样品也表现出良好的检测性能,证明其具有良好的临床实用性和在复杂样本中检测微量PTEN ^(R130*)突变的潜能。An analytical method based on amplification refractory mutation system-real time fluorescence-quantitative polymerase chain reaction(ARMS-qPCR)was developed for detection of gene mutation PTEN^(R130*) mutation in thyroid cancer.Allele-specific and reference primers for the PTEN^(R130*) mutation were designed for allele-specific and reference amplification of each sample at the same time,and theΔCt value was obtained by Ct-allele-specific value minus Ct-reference value to analyze the variant allele frequency(VAF).The ARMS-qPCR method could detect VAF as low as 0.01%,with a linear range of 0.1%to 90%VAF,and could identify PTEN mutations in genomic DNA as low as 4 copies.This method successfully detected 5 cases of mutation with VAF>0.1%in 24 thyroid tumor samples.In addition,the ARMS-qPCR method also showed good detection performance for PTEN^(R130*) mutant spiked plasma samples under the electrochemical enrichment,indicating clinical practicability and the potential to detect trace PTEN^(R130*) mutation in complex samples.

关 键 词：PTEN^(R130*)突变 甲状腺癌 突变扩增系统-实时荧光定量聚合酶链式反应 

分 类 号：R736.1[医药卫生—肿瘤]