猪传染性胃肠炎病毒实时荧光定量PCR检测方法的建立与应用  

Establishment and application of a fluorescent quantitative PCR for detection of transmissible gastroenteritis virus of swine

作　　者：张雪[1,2] 杨倩[1] 于红欣[1] 王俊[1] 孟凡伟[1] 周双海[1] 

机构地区：[1]北京农学院动物科学技术学院,北京102206 [2]北京市怀柔区动物疫病预防控制中心,北京101400

出　　处：《中国兽医科学》2015年第6期644-648,共5页Chinese Veterinary Science

基　　金：天津市宁河原种猪场科技计划项目(2011)

摘　　要：为了建立一种定量检测猪传染性胃肠炎病毒(TGEV)的实时荧光定量PCR方法,用RT-PCR扩增TGEV的M基因片段,构建含有TGEV M基因片段的重组质粒。用系列稀释后的重组质粒为模板,基于SYBR GreenⅠ来进行检测TGEV的实时荧光定量PCR扩增。结果显示,扩增效率为103.0%,相邻扩增曲线之间间距均匀,所有产物的熔解曲线具有单一整齐的峰,标准曲线具有优良的线性关系,最低可准确检测63copies/μL的核酸模板,重复性试验的变异系数小于3%。用建立的PCR对3种常见猪源RNA病毒的检测结果均为阴性,对65份临床样品的检出率高于常规PCR。结果表明,建立了一种灵敏度高、特异性强、重复性好的检测TGEV的SYBR GreenⅠ实时荧光定量PCR方法,可用于TGEV的定量检测和猪传染性胃肠炎的早期快速诊断。The objective of this study was to develop a real-time fluorescent quantitative PCR(FQPCR)assay for quantitative detection of transmissible gastroenteritis virus of swine(TGEV).A fragment of M gene in TGEV was amplified by RT-PCR method,and the recombinant plasmid contained the M gene fragment was constructed.The recombinant plasmid was diluted serially as templates and a real-time FQPCR amplification for detection of TGEV based on SYBR GreenⅠ was performed.The results showed that the amplification efficiency was 103.0%,all the distances between the adjacent amplification curves were even,all the melting curves of products showed only one smooth peak,and the standard curve was with excellent linear relationship.The assay had an accurate detectable limit of 63copies/μL,and its coefficient of variation was less than 3%in the reproducible assays.No amplification was detected by this method from three kinds of epidemic swine RNA virus samples.The detectable rate of 65 clinical samples with this method was more than conventional PCR.These research data revealed that a high specific,sensitive and reproducible SYBR GreenⅠreal-time FQ-PCR for determination of TGEV was established,which can be used for the quantitative determination of TGEV and the early and quick diagnosis of transmissible gastroenteritis of swine.

关 键 词：猪传染性胃肠炎病毒 SYBR GreenⅠ 荧光定量PCR 早期快速诊断 

分 类 号：S852.65[农业科学—基础兽医学]