猪诺如病毒巢式RT－PCR检测方法的建立与应用

摘要

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.%为了解中国猪群中猪诺如病毒感染情况，本研究根据猪诺如病毒保守的RNA依赖性RNA多聚酶（ RNA Dependent RNA Polymerase，RDRP）基因序列设计了一套巢式RT－PCR引物，用该引物从猪粪便样品中扩增出与预期目的产物大小一致的条带，经克隆、测序后获得的序列与GenBank数据库序列比对，证明所扩增的序列属于猪诺如病毒，表明本研究成功建立了猪诺如病毒巢式RT－PCR检测方法。该检测方法具有高特异性和敏感性，应用该方法检测某猪场健康育肥猪粪便样品，阳性率为54．5％。通过RDRP基因进行核苷酸同源比对发现本文获得的猪诺如病毒Wuning 1株属于GII群诺如病毒，与美国猪诺如病毒毒株OH－QW101／03／US、中国猪诺如病毒毒株Norovirus pig／GII／Ch6／China／2009核苷酸序列同源性最近。值得注意的是猪诺如病毒Wun⁃ing1毒株与武汉人诺如病毒毒株E2120／CHN／2010核苷酸同源性高达77．1％，说明猪诺如病毒Wuning 1毒株与武汉人诺如病毒基因组上亲缘性较近。本研究成功建立了一种检测猪诺如病毒的巢式RT⁃PCR方法，并应用于临床猪粪便样品的检测，为猪诺如病毒的诊断与监测提供了可供借鉴的方法、为猪诺如病毒的进化、分子病毒学及与人诺如病毒关系的深入研究提供依据。