Detection of Human Rotaviruses in Fresh and Estuarine Waters by Dot-blot Hybridization

Abstract

This study was conducted to evaluate the suitability of dot-blot hydridization for detecting rotavirus in fresh and estuarine water samples using cloned DNA of genome segment 6 of human strain Wa and simian SA-11 rotaviruses. Probes of Wa and SA-11 cross-hybridized with heterologous RNAs of SA-11 and Wa rotaviruses under low stringency conditions. However, cross-hybridization reactions were minimized or eliminated under high stringency conditions, while homologous reactions were not affected. Wa probe did not react with representatives of the major enterovirus groups or hepatitis A virus. Hybridization detection sensitivity was not affected by organic material present in waters or proteinaceous material of eluents. Hybridization using cDNA of Wa rotavirus was found to be more sensitive than either Rotazyme ELISA or electron microscopy for detecting rotavirus in stool specimens. Dot-blot hybridization using cDNA of genome segment 6 of Wa rotavirus was found to be more sensitive than SPRIA for detecting rotavirus in estuarine and fresh water samples. Only 9 out of 54 (18.5%) estuarine and fresh water samples were positive by SPRIA, whereas dot-blot hybridization with Wa probe resulted in 21 out of 54 (38.8%) positive samples. One out of 20 (5%) stool samples and 23 out of 54 (44.6%) estuarine and fresh water samples were found to be positive by a pBR-322 plasmid probe. These results indicate that pBR-322 and related plasmids are present in high frequency in fecally polluted waters. Although different numbers of positive results were found with Wa and pBR-322 probes, more research is required to ascertain that hybridization reactions with such plasmids do not occur when using cDNA rotavirus probes.