江西农业大学学报2016，Vol.38Issue(2)：338-346,9.DOI:10.13836/j.jjau.2016048
猪诺如病毒巢式RT－PCR检测方法的建立与应用
Development and Application of a Nested RT-PCR Assay for Detection of Porcine Norovirus
摘要
Abstract
The objective in of study was to establish a nested reverse transcription⁃polymerase chain reac⁃tion (RT⁃PCR) for the detection of porcine noroviruses in Jiangxi,China.For this purpose,two pairs of primers specific to RNA Dependent RNA Polymerase (RDRP) gene were designed and used to amplify the fragments of porcine noroviruses.The amplicons from the RT⁃PCR established were sequenced after cloning.Fragments we obtained turned out to be partial RDRP gene of porcine norovirus after BLAST Searching in NCBI⁃GenBank. The results indicated that we successfully established a nested RT⁃PCR assay for porcine norovirus detection and it was only able to amplify porcine norovirus with sensibility of 1.0 ´101copies/μL.The established nested RT⁃PCR assay was applied to detect fecal samples from asymptomatic fattening pigs from a farm and the posi⁃tive rate was 54. 5%. The results of nucleotide sequence identity and phylogenetic analyses demonstrated that Wuning 1 strain had a close relationship with OH⁃QW101/03/US strain and pig/GII/Ch6/China/2009 strain. And 77.1%nucleotide sequence identity between Wuning 1 and Hu /Wuhan/E2120/CHN/2010 strain indica⁃ted a genetically closed relationship between porcine and human noroviruses.To our knowledge,this is the first report on nested RT⁃PCR assay for detection of Porcine norovirus,which will provide a valuable tool for further studies in term of norovirus molecular virology as a potential zoonotic pathogen.The establishment of nested RT⁃PCR for PNoV detection revealed the genetic characteristics of porcine norovirus identified in this study and the relationship between Porcine norovirus and human norovirus.关键词
猪诺如病毒/巢式RT-PCR/同源性分析/系统进化树分析Key words
porcine norovirus/nested RT-PCR/homology analysis/phylogenetic analysis分类
农业科技引用本文复制引用
陈燕君,叶昱,吴琼,唐玉新,周信荣,宋德平,黄瑫,彭棋,李安琪,张帆帆,张田生..猪诺如病毒巢式RT－PCR检测方法的建立与应用[J].江西农业大学学报,2016,38(2):338-346,9.基金项目
国家自然科学基金项目（31260611，31372457）、江西省研究生创新专项资金项目（YC2014-S181）、江西省自然科学基金项目（2013BAB204017）和江西省落地计划（2013BAB204017） Project supported by the National Nature Science Foundation of China （31260611，31372457），Funding from the Innovation Fund Designated for Graduate Students of Jiangxi Province （ YC2014-S181），Nature Science Founda-tion of Jiangxi Province，China （2013BAB204017） and the Landing Program Fund of Jiangxi Province，China （）
