猪札幌病毒TaqMan荧光定量RT-PCR检测方法的建立及初步应用  

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

作　　者：陈琰[1] 沈权[1] 杨世兴[1] 康雁君[1] 华修国[1] 

机构地区：[1]上海交通大学农业与生物学院,上海200240

出　　处：《畜牧兽医学报》2010年第2期246-250,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

摘　　要：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。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 GⅢ,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.

关 键 词：猪札幌病毒 TAQMAN探针 荧光定量RT-PCR 

分 类 号：S854.43[农业科学—临床兽医学] S852.659.6[农业科学—兽医学]