猪细小病毒与猪伪狂犬病毒二重SYBR GreenⅠ实时荧光PCR方法的建立与检测  

Simultaneous detection of porcine pseudorabies virus and porcine parvovirus by duplex real-time PCR

作　　者：郑兰兰[1,2] 金钺[1] 李明凤[2] 郭显坡[1,2] 陈红英[1,2] 崔保安[1,2] 魏战勇[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《中国兽医学报》2014年第5期690-694,共5页Chinese Journal of Veterinary Science

基　　金：河南省重大科技攻关项目(111100110300)

摘　　要：本研究旨在建立一种能快速、灵敏、同时检测出猪细小病毒与猪伪狂犬病毒的基于SYBR GreenⅠ实时荧光定量PCR方法。参照GenBank中登录的相关基因序列,设计了2对引物分别用于扩增PRV gH基因与PPV NS1基因的部分片段。将测序正确的PRV gH基因与PPV NS1基因片段克隆入pGEM-T Easy载体,转化大肠杆菌DH5α,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBR GreenⅠ荧光定量PCR标准曲线和熔解曲线,并对其灵敏性、特异性和重复性进行验证。结果表明,猪细小病毒与猪伪狂犬病毒荧光定量PCR的标准曲线的Tm值分别为80.9℃和86.5℃,熔解曲线特异,灵敏度分别可达248拷贝/μL和160拷贝/μL,是普通PCR检测方法的100倍。本次建立的猪细小病毒与猪伪狂犬病毒荧光定量PCR检测方法实现了2种病毒的同时检测,能够对PRV、PPV混合感染的临床病料进行快速诊断。The aim of this study was to develop a SYBR Green I real-time quantitative PCR for detecting porcine pseudorabies virus （PRV） and porcine parvovirus （PPV） simultaneously, quickly and flexibly. According to genome sequences with gH of PRV and NS1 of PPV published in GenBank,two pairs of primers were designed. The fragments of gH and NS1 gene were amplified with traditional PCR. The PCR products were cloned into pGEM-T vector and sequenced. The positive recombinant plasmids were used as quantita- tive templates to generate standard curve and melt curve. Sensitivity assay, reproducibility of the assay and specificity assay were determined. The results demonstrated that standard curve established by recombinant plasmids were specific,the Tm of PPV was 80. 9℃ and the Tm of PRV was 86. 5℃. The detection limit of real-time PCR was 248 copies per 9L for PPV, and 160 copies per μL for PRV. The quantitative PCR was more reproductive and specific than traditional PCR. These results indicated that the SYBR Green I fluorescent quantitative PCR for detecting PPV and PRV were developed for the early, simultaneous and rapid detection and analyzing the infect degree of PPV and PRV quantitatively.

关 键 词：猪细小病毒 猪伪狂犬病毒 SYBR GreenⅠ 

分 类 号：S852.65[农业科学—基础兽医学]