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PTENR130*突变

魏佳 王瑶琪 王振新 孟宪瑛

引用本文: 魏佳, 王瑶琪, 王振新, 孟宪瑛. 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PTENR130*突变[J]. 分析化学, 2022, 50(5): 701-710. doi: 10.19756/j.issn.0253-3820.221008 shu
Citation:  WEI Jia,  WANG Yao-Qi,  WANG Zhen-Xin,  MENG Xian-Ying. Highly Sensitive Detection of PTENR130* Mutation in Thyroid Cancer Based on Amplification Refractory Mutation System-Real Time Fluorescence-Quantitative Polymerase Chain Reaction[J]. Chinese Journal of Analytical Chemistry, 2022, 50(5): 701-710. doi: 10.19756/j.issn.0253-3820.221008 shu

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PTENR130*突变

    通讯作者: 王振新,E-mail:wangzx@ciac.ac.cn; 孟宪瑛,E-mail:mengxiany@jlu.edu.cn
  • 基金项目:

    吉林省科技发展计划项目(No.20210204049YY)和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PTENR130*突变。针对PTENR130*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PTENR130*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PTENR130*突变。此外,ARMS-qPCR法对电化学富集后的PTENR130*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PTENR130*突变的潜能。

English


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