Detection of field isolates of human and animal group C rotavirus by reverse transcription-polymerase chain reaction and digoxigenin-labeled oligonucleotide probes

Abstract

Rotaviruses (RV) are important etiological agents of acute gastroenteritis in infants and young children, as well as the young of a variety of animals worldwide. These viruses belong to Reoviridae family and contain a genome of 11 segments of double-stranded RNA (dsRNA). Two major proteins, VP4 and VP7, encoded by genome segments 4 and 7, 8 or 9, respectively, evoke a neutralizing antibody response and form the basis for the current classification of group (gp) A rotavirus into P (VP4) and G (VP7) serotypes. Although much recent progress has been made on the molecular biology of gp C RV, routine methods to detect and discriminate human, porcine, and bovine strains are not available widely. In this study, a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) and digoxigenin-labeled (dig) oligonucleotide probes using chemiluminescence has been developed to detect and discriminate VP7 genes from culture-adapted and field isolates of human, porcine and bovine gp C RV. The multiplex RT-PCR and dig-probes were specific for the VP7 genes of human, porcine and bovine gp C RV and allowed detection and characterization of single and mixed infections of porcine gp C RV with porcine gp A or gp B rotaviruses. Detection rates for gp C RV were more than 50% when compared with polyacrylamide gel electrophoresis. These new diagnostic assays may help determine the epidemiological importance of these viruses in human and animal infections.