TaqMan探针实时PCR检测人MTHFR基因C677T多态性方法的建立  

Establishment of TaqMan Probe Real-time PCR for Detecting MTHFR C677T Polymorphism

作　　者：王苏梅 王克华[2] 魏斌 于建春 盖凌 董云玲 吕雪梅 刘锦云 

机构地区：[1]山东省计划生育科学技术研究所山东省优生技术重点实验室,济南250002 [2]山东中医药大学附属医院,济南250012

出　　处：《中国优生与遗传杂志》2012年第9期11-14,共4页Chinese Journal of Birth Health & Heredity

基　　金：山东省计划生育科技发展项目

摘　　要：目的建立TaqMan探针实时PCR检测人MTHFR基因C677T多态性的方法。方法设计一对MTHFR基因C677T多态位点的引物及TaqMan探针,采用TaqMan探针实时PCR扩增SNP分型方法检测唇腭裂患者及其父母共100人的MTHFR基因C677T多态性,与常规PCR-RFLP方法进行一致率比较,并对其特异性、敏感性和重复性以及成本-效益等进行评价,同时对部分实时PCR产物样本进行测序验证。结果运用TaqMan探针实时荧光PCR技术对MTHFR基因C677T多态性检测结果准确,特异性好,与常规PCR-RFLP方法结果具有高度一致性,Kappa=0.922>0.75(P=0.000);检测灵敏度可达2×103拷贝;重复性好、高通量、无污染、安全性好;随机样品TaqMan探针分型结果与测序结果完全一致。结论成功建立了TaqMan探针实时PCR检测人MTHFR基因C677T多态性的方法;此方法是常规临床诊断及大规模群体研究的良好平台。Objective： To establish the approach of TaqMan probe real - time PCR for detecting MTHFR C677T polymorphism. Methods ： A pair of primers and TaqMan probe were designed in order to detect MTHFR C677T polymorphism of nonsyndromic cleft lip with or without cleft palate （NSCL/P） patients and their parents （100 people） by post -PCR read SNP typing based on real - time PCR amplification comparing with traditional PCR - RFLP. The specificity, sensitivity, repetitiveness and cost - benefit of TaqMan probe real -time PCR were evaluated, and the PCR products were also subjected to gene sequence analysis to validate the results of TaqMan probe real - time PCR. Results ： Detection of MTHFR C677T polymorphism was specificly and accurately finished by TaqMan probe real - time PCR, and the results were highly consistent with that of traditional PCR - RFLP. Kappa = 0. 922 〉 0. 75 （ P = 0. 000）, The sensitivity of detection reaches 2 x 103 copy and the TaqMan probe real- time PCR is highly repetitive, high -through- put, pollution - free and safe. The results of random sample in TaqMan probe typing are consistent with the results of gene sequence analysis. Conclusion ： The approach of TaqMan probe real - time PCR for detecting MTHFR C677T polymorphism is successfully established, providing a favorable platform for conventional clinic diagnosis and epidemiological study.

关 键 词：TAQMAN探针 实时荧光PCR 单核苷酸多态性 亚甲基四氢叶酸还原酶 C677T突变 聚合酶链反应-限制性内切酶片段长度多态性 非综合征性唇腭裂 

分 类 号：R440[医药卫生—诊断学]