猪札幌病毒TaqMan荧光定量RT-PCR检测方法的建立及初步应用

根据猪札幌病毒(Porcine Sapovirus,SaV)的VP1保守基因序列设计引物和探针,通过对荧光定量RT-PCR反应条件的优化,建立了TaqMan荧光定量RT-PCR检测方法,并与常规RT-PCR检测方法进行了比较。结果表明,荧光定量RT-PCR方法的检测灵敏度可达16.1拷贝.μL-1,而常规RT-PCR方法的灵敏度为1.61×103拷贝.μL-1。对216份粪样的检测结果进一步表明该法(检出4份)比常规RT-PCR方法(检出3份)的灵敏度高。系统进化分析表明,该4株病毒均为GⅢ型,与SaV上海分离株(FJ387164)同源性为100%。该方法具有灵敏度高、特异性强、操作简便等优点,适合于猪SaV感染的流行病学调查和临床诊断。

Development and Preliminary Application of TaqMan Fluorescence Quantitative RT-PCR Assay for Detection of Porcine Sapovirus

The primers and probes were designed and synthesized according to the conserved VP1 sequences of porcine Sapovirus (SaV), and a TaqMan fluorescence quantitative RT-PCR assay were developed by optimizing the reaction conditions. Results showed that the fluorescence quantitative RT-PCR assay could detect 16.1 copies·μL~(-1) of plasmid DNA, while the sensitivity of the routine RT-PCR was 1.61×10~3 copies·μL~(-1). 216 stool samples were then detected by the established quantitative RT-PCR assay, and the results were compared with that of routine RT-PCR. It also showed that the sensitivity of established method was higher than that of the routine RT-PCR. Phylogenetic analysis indicated that all of the 4 SaV strains we had identified belonged to G Ⅲ, and shared 100% nucleotide homology with another Shanghai porcine SaV strain (FJ387164). The TaqMan fluorescence quantitative PCR assay, which is more specific, sensitive and accurate, can be used for the epidemiological investigation and diagnosis of porcine SaV infection.