Hybridization probes for the detection and differentiation of two serotypes of porcine rotavirus

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.