猪伪狂犬病毒与猪圆环病毒2型二重SYBR GreenⅠ实时荧光PCR检测方法的建立

Simultaneous detection of porcine pseudorabies virus and porcine circovirus 2 by duplex real-time PCR

参照GenBank登录的相关基因序列,设计了2对引物分别用于扩增伪狂犬病毒(PRV)gH基因与猪圆环病毒2型(PCV2)ORF2基因的部分片段。将测序正确的PRV gH基因与PCV2ORF2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。结果显示,PCV2与PRV荧光定量PCR的标准曲线的Tm值分别为80.8℃和86.7℃,熔解曲线特异,灵敏度分别可达215拷贝/μL和180拷贝/μL,是普通PCR检测方法的100倍。结果表明,建立的PCV2与PRV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、PCV2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 quanntative PCR for de- tecting porcine pseudorabies virus（PRV） and porcine circovirus 2 （PCV2） simultaneously,quickly and flexibly. According to genome sequences within gH of PRV and ORF2 of PCV2 published in GenBank,two pairs Of primers were designed. The fragments of gH and ORF2 gene were ampli- fied with traditional PCR. The PCR products were cloned into pGEM-T vector and sequenced. The positive recombinant plasmids were used as quantitative templates to generate standard curve and melt curve. Sensitivity,reproducibility and specificity of the method were determined. The re- suits demonstrated that standard curve established by recombinant plasmids were specific,the Tm of PCV2 was 80.8 ℃and the Tm of PRV was 86.7℃ The detection limit of real-time PCR was 215 copies/μ for PCV2,and 180 copies/μL for PRV. The quantitative PCR was higher reproduc- tive and specific than traditional PCR. These results indicated that the developed SYBR Green l fluorescent quantitative PCR for detecting PCV2 and PRV lay a basis of the early.simultaneous and rapid detection and analyzing the infect degree of PCV2 and PRV quantitatively.