VP4 gene specific RT-PCR for detection of bovine group A rotaviruses

RT-PCR assay was standardised with partial length primers specific to VP4 g ene of bovine group A rotaviruses. The double stranded RNA (dsRNA) was extr acted from MA 104 cell culture grown prototype rotaviruses by GIT lysis met hod. The presence of dsRNA in the extracts was confirmed by RNA-polyacrylam ide gel electrophoresis (RNA-PAGE) with silver staining. To develop the RT- PCR assay, dsRNA was first converted into complimentary DNA (cDNA) by rever se transcriptase enzyme and subsequently amplified by Taq DNA polymerase. T he oligonucleotide primers used in RT-PCR assay were specific to both 3' an d 5' ends of VP4 gene, which is highly, conserved among group A rotaviruses . The presence of expected 876 bp long PCR product was visualised by agaros e gel electrophores-is. The primer pair used in present study specifically amplified VP4 gene. The results of present study suggested that RT-PCR assa y could be used for detection of rotaviruses.