PRRSV、PCV2与CSFV多重SYBRGreenⅠ实时荧光PCR检测方法的建立与初步应用  

Simultaneous detection of Porcine reproductive and respiratory syndrome virus,porcine circovirus 2 and classical swine fever virus by multiplex real-time PCR

作　　者：高晓平[1] 郑兰兰[2,3] 于朋飞[2,3] 韩志涛[2,3] 陈红英[2,3] 崔保安[2,3] 魏战勇[2,3] 

机构地区：[1]河南农业大学食品科学技术学院,河南郑州450002 [2]河南农业大学牧医工程学院,河南郑州450002 [3]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》2015年第9期1409-1414,1428,共7页Chinese Journal of Veterinary Science

基　　金：河南省重大科技攻关资助项目(111100110300)

摘　　要：旨在建立一种能快速、灵敏、同时检测出猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)与猪瘟病毒(CSFV)3种病毒的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3对引物分别用于扩增PRRSV ORF7基因、PCV2ORF2基因与CSFV 5′端保守序列的部分片段。将测序正确的3段基因片段分别克隆入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标准曲线和溶解曲线,并对其灵敏性、特异性和重复性进行验证。结果表明,PCV2、PRRSV与CSFV荧光定量PCR的标准曲线的Tm值分别为84.6、86.7、89.3℃,溶解曲线特异,灵敏度分别可达181、202、177拷贝/μL,是普通PCR检测方法的100倍。本试验建立的PRRSV、PCV2与CSFV的荧光定量PCR检测方法实现了3种病毒的同时检测,能够对PRRSV、PCV2、CSFV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 de- tectingporcine reproductive and respiratory syndrome virus （PRRSV）, porcine circovirus 2 （PCV2） and classical swine fever virus（CSFV） simultaneously,quickly and flexibly. According to genome sequences within ORF7 of PRRSV,ORF2 of PCV2 and the 5＇conserved sequence of CSFV published in GenBank, three pairs of primers were designed. The fragments of the three genes were amplified with traditional PCR. The PCR products were cloned into pGEM-T vector and se- quenced. The positive recombinant plasmids were used as quantitative templates to generate stand- ard 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 PCV2 was 84.6℃ ,the Tm of PRRSV was 86.7℃ and the Tm of CSFV was 89.3℃. The detection limit of real-time PCR was 181 copies per μL for PCV2,202 copies perμL for PRV and 177 copies perμL for CSFV. The quantitative PCR was more reproductive andspecific than traditional PCR. These results indicated that the SYBR Green I fluorescent quantita- tive PCR for detecting PCV2, CSFV and PRRSV were developed. It lays the basis of the early, sim- ultaneous and rapid detecting and analyzing the infect degree of PCV2,CSFV and PRRSV quanti- tatively.

关 键 词：猪繁殖与呼吸综合征病毒 猪圆环病毒2型 猪瘟病毒 SYBR GreenⅠ 

分 类 号：S852.65[农业科学—基础兽医学]