牛诺如病毒和牛环曲病毒双重PCR检测方法的建立及初步应用

为建立能同时检测牛诺如病毒(BNoV)和牛环曲病毒(BToV)的方法,根据BNoV和BToV基因组的保守区域设计2对特异性引物,预期特异性扩增BNoV和BToV的片段大小分别为542,698 bp,建立了BNoV和BToV的双重PCR检测方法。特异性检测结果显示,该方法能特异性扩增BNoV和BToV的目的条带,且未扩增出其他犊牛腹泻病原;对BNoV和BToV的最低检出量分别为1.90×10^4,1.86×10^4拷贝/μL。利用该方法对采自河南部分地区的184份犊牛腹泻样品的检测结果显示,BNoV和BToV的阳性检出率分别为11.41%和12.50%,与单一PCR检测结果一致。结果表明,本试验建立的方法对BNoV和BToV的鉴别诊断、混合感染和流行病学调查具有重要的应用价值。

Establishment and preliminary application of a duplex PCR for detection of BNoV and BToV

To establish a PCR method for simultaneous detection of bovine norovirus(BNoV) and bovine torovirus(BToV),two pairs of specific primers were designed based on the conserved regions of BNoV and BToV genomes.The assay was highly specific to amplify the fragments of 542 bp for BNoV and 698 bp for BToV,respectively,and had no amplification for other related viruses.The sensitivity of the method was 1.90×10^4 copies/μL for BNoV and 1.89×10^4 copies/μL for BToV,as well as a good specificity.Furthermore,this duplex PCR method was used to test 184 clinical samples that collected from the diarrheic calves in Henan province.The positive rates of BNoV and BToV were 11.41% and 12.50%,respectively,which were consistent with single PCR results.Therefore,the duplex PCR method has a potential application in clinical diagnosis,differential diagnosis in mixed infections and epidemiological investigation of BNoV and BToV.