检测伪狂犬病毒gB基因荧光定量PCR方法的建立

利用DNAMAN软件对GenBank登录的伪狂犬病病毒（PRV）各毒株gB基因序列进行比对分析,选择其保守区域设计合成特异性的引物和TaqMan探针,同时利用普通PCR技术扩增获得全长的伪狂犬病毒gB基因,并克隆到pMD20-T载体上作为阳性标准品。通过对荧光定量PCR反应条件的优化,建立了一种快速检测伪狂犬病病毒的荧光定量PCR技术。该检测技术具有较高的灵敏性、特异性和可靠性。对制备的pMD-gB阳性标准品的检测结果表明,所建立的伪狂犬病毒TaqMan荧光定量PCR最低检测极限可达1.50×102拷贝/反应;同时相比于普通PCR方法其灵敏度高100～1 000倍以上,并且重复性好。在对60份临床样品的检测中,荧光定量PCR不仅检出了普通PCR检测为阳性的样品,且检出了2份普通PCR未检出的样品,进一步证实了该方法快速、灵敏性好,可用于PRV感染的早期快速定量检测和肉类食品进出口检疫。

Development of fluorescence quantitative PCR assay to detect gB gene of pseudorabies virus

Pseudorabies virus（PRV） gB gene sequences including several genotypes strains available in GenBank were aligned with the DNAMAN software,the conserved region was then subjected to design specific primers and TaqMan probe.Then full-length PRV-gB gene amplified by PCR was cloned into pMD20-T vector and it was used as positive standard.A rapid fluorescence quantitative PCR detection method of pseudorabies virus was established by optimizing reaction conditions.The detection method had high specificity and reliability.The detection limit of FQ-PCR was 1.50×102 copies per reaction in analyzing pMD-gB standard.Compared with conventional PCR for diagnosis of PRV,the sensitivity of FQ-PCR method was 100 to 1 000-fold more than the conventional PCR and with good reproducibility.The detection results of 60 clinical specimens indicated that FQ-PCR not only checked out the positive samples which did the same with conventional PCR,but also checked out othed 2 positive samples which did not with conventional PCR.It confirmed that the method is sensitive.This assay can be used for early rapid detection of PRV,and for quarantine of import and export of meat products.