牦牛星状病毒的RT-PCR检测方法的建立和变异分析

Development of an RT-PCR Assay and Variation Analysis for Yak Astrovirus

近期作者实验室从腹泻牦牛粪便样本中鉴定出一种新型星状病毒(AstV)——牦牛AstV,本研究旨在建立检测牦牛AstV的RT-PCR方法。首先设计引物扩增牦牛AstV 630bp的ORF2基因片段,进行序列分析;在此基础上设计检测引物,建立检测牦牛AstV的RT-PCR方法。结果显示,从20份牦牛腹泻粪便中扩增出13个牦牛AstVORF2基因片段,核苷酸相似性为99.3%~100%,而与牛肠源星状病毒(BAstV)ORF2基因的相似性仅为59.5%~80.8%;所建立的RT-PCR方法只扩增牦牛AstV的特异片段,对BAstV和其他无关病原无扩增;对病毒核酸的最低检测限为57.3fg·μL^(-1);比较试验表明,所建立的RT-PCR方法对牦牛AstV的检出率明显优于现有检测BAstV的RT-PCR方法。其对牦牛腹泻与健康粪便样本中牦牛AstV的检出率分别为55.5%(76/137)和7.7%(2/26)(P<0.01)。试验结果表明,扩增的ORF2基因片段的核苷酸序列在牦牛AstV毒株间高度保守,但相对于BAstV变异大;基于该序列建立的检测牦牛AstV的RT-PCR方法特异性好,灵敏度高,稳定性好,为牦牛AstV的检测和流行病学调查提供了有力工具;同时,本试验结果也提示牦牛AstV与犊牦牛腹泻有联系。

Recently,our laboratory identified a novel astrovirus from fecal samples of diarrheic yak,the aim of this study was to develop an RT-PCR assay for detecting the novel yak astrovirus（yak AstV）.Firstly,one pair primers were designed to amplify ORF2 gene fragments（630bp in length）of yak AstV.By sequence analysis,one pair primers of RT-PCR assay were then designed for detecting yak AstV.We obtained 13 ORF2 gene fragments from 20 fecal samples of diarrheic yak,which shared a homology of 99.3%-100% within fragments,but only a homology of 59.5%-80.8% with bovine astrovirus（BAstV）.The RT-PCR assay developed in this study was specific to yak astrovirus,no amplifying for BAstV and other pathogens tested in this study.The detection limit of viral nucleic acid of the assay was 57.3fg·μL^-1.The assay was significantly better in detecting yak AstV compared to the other two reported assays for detecting bovine astrovirus.The detecting results of clinical samples by this assay showed that the positive rates of yak AstV were55.5%（76/137）in diarrheic fecal samples and 7.7%（2/26）in healthy fecal samples.Compared to healthy samples,yak AstV was significantly more prevalent in diarrheic fecal samples（P0.01）.In conclusion,ORF2 gene sequences of yak AstV cloned in this study varied a lot compared with those of BAstV,but were highly conserved within yak AstV species.Thus,the RT-PCR for detecting yak AstV based on the ORF2 sequences of yak AstV greatly improved the detection rate of yak AstV,providing a useful tool for detection of this novel virus and epidemiological investigation.Meanwhile,the results of this study indicated that yak AstV might be associated with diarrhea in yak calves.