Rosen B I, Saif L J, Jackwood D J, Gorziglia M
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Ohio State University, Wooster 44691.
Vet Microbiol. 1990 Sep;24(3-4):327-39. doi: 10.1016/0378-1135(90)90181-t.
A dot hybridization assay was developed to detect and differentiate two serotypes of porcine rotavirus by hybridization of specific probes to rotavirus RNA dotted onto nylon membranes. The assay was conducted using radiolabeled probes prepared from cDNA clones of OSU (serotype 5) and Gottfried (serotype 4) rotavirus gene 9 segments. The probes were prepared by excision of full length cDNA copies of gene 9 inserts from recombinant plasmids followed by nick translation and 32P-dCTP labeling. Rotavirus RNA samples used in the assay were prepared from tissue culture, and intestinal contents from gnotobiotic and conventional pigs. Optimal conditions for hybridization consisted of 52 degrees C, 50% formamide, and 5 x SSC. Hybridization studies with the OSU and Gottfried gene 9 probes have shown them to be specific with only low levels of cross-reactivity with heterologous rotavirus preparations. Sensitivity studies have demonstrated the ability of the probes to detect at least 2 ng of rotavirus RNA. Further development and refinement of this technique may provide a useful tool for the detection and differentiation of porcine rotavirus serotypes.
开发了一种斑点杂交试验，通过将特异性探针与点样在尼龙膜上的轮状病毒RNA杂交，来检测和区分猪轮状病毒的两种血清型。该试验使用从OSU（血清型5）和Gottfried（血清型4）轮状病毒基因9片段的cDNA克隆制备的放射性标记探针进行。探针通过从重组质粒中切除基因9插入片段的全长cDNA拷贝，然后进行缺口平移和32P-dCTP标记来制备。试验中使用的轮状病毒RNA样本取自组织培养物，以及无菌猪和普通猪的肠道内容物。杂交的最佳条件为52℃、50%甲酰胺和5×SSC。用OSU和Gottfried基因9探针进行的杂交研究表明，它们具有特异性，与异源轮状病毒制剂的交叉反应水平很低。敏感性研究证明，这些探针能够检测到至少2 ng的轮状病毒RNA。该技术的进一步开发和完善可能为猪轮状病毒血清型的检测和区分提供一种有用的工具。
