Parwani A V, Rosen B I, Flores J, McCrae M A, Gorziglia M, Saif L J
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Ohio State University, Wooster 44691.
J Vet Diagn Invest. 1992 Apr;4(2):148-58. doi: 10.1177/104063879200400206.
Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Crocker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.
斑点杂交和Northern杂交分析方法得以开发，用于使用放射性标记的血清型6（内布拉斯加小牛腹泻病毒[NCDV]和英国[UK]毒株）或血清型10（克罗克[Cr]毒株）VP7基因探针来检测和区分A组牛轮状病毒血清型。使用克隆的VP7基因（NCDV和UK毒株）或逆转录的mRNA（Cr毒株）作为模板，通过聚合酶链反应（PCR）生成包含主要序列多样性区域的部分长度VP7特异性cDNA。对由PCR生成的cDNA制备的放射性标记探针在各种严谨条件下进行测试，以优化杂交分析。在高严谨条件（52°C、50%甲酰胺、5×标准柠檬酸盐缓冲液）下，NCDV、UK和Cr探针可血清型区分从细胞培养繁殖病毒制备的RNA样本或感染的无菌小牛粪便标本中的牛轮状病毒分离株。在斑点杂交分析中对NCDV和Cr VP7探针的敏感性和特异性进行了表征，估计这些探针可检测至少1 ng的病毒RNA。使用VP7探针获得的血清型鉴定结果与使用血清学检测获得的结果相似。这些分析方法的进一步开发可能为快速检测和区分田间小牛粪便样本中的牛轮状病毒血清型提供一种有用的手段。
