猪源伪狂犬病病毒gB基因SYBR GreenⅠ实时荧光定量PCR检测方法的建立

Development of a SYBR GreenⅠreal-time PCR for detection of gB gene of pseudorabies virus in swine

为加强对猪源伪狂犬病病毒（pseudorabies virus,PRV）的监测,在PRV gB基因的保守区设计特异性引物,并建立了检测猪源PRV的实时定量PCR。该方法可检测PRV的最低TCID50为2×10^1/mL,比伪狂犬病诊断技术国家标准中的PCR方法敏感100-1 000倍,且特异性、重复性好。利用该方法测定了PRV SC株在感染Vero细胞后的复制动态;结果显示,PRV在感染后第12小时进入复制高峰期,在感染后第24小时达到平台期。本方法具有快速、敏感和重复性好等特点,能够为研究病毒的复制动态或临床样品的检测提供技术支持。

In order to detect pseudorabies virus（PRV）in swine,we designed a pair of specific primers for the conservative region of PRV gB gene,and a real-time PCR was developed.The method had a good linear relationship within the scope of the 1×10^2 to 1×10^7 copies/mL.The lowest detectable concentration was up to 2×10^1/mL in TCID50 and its sensitivity was 100-to 1 000-fold more than the conventional PCR.The method was used to determinate the replication dynamics of PRV SC strains on Vero cells.The results showed that the logarithmic phase of SC strains was appeared at 12hpost-infection and reached the plateau phase at 24hpost-infection.This method was rapid,sensitive,specific and accurate,and it would provide a technical support for the study of viral replication dynamics and the clinical sample detection.