VP 4 gene specific RT-PCR for detection of bovine group A rotaviruses

Abstract

RT-PCR assay was standardised with partial length primers specific to VP₄ gene of bovine group A rotaviruses. The double stranded RNA (dsRNA) was extracted from MA 104 cell culture grown prototype rotaviruses by GIT lysis method. The presence of dsRNA in the extracts was confirmed by RNA-polyacrylamide gel electrophoresis (RNA-PAGE) with silver staining. To develop the RT-PCR assay dsRNA was first converted into complimentary DNA (cDNA) by reverse transcriptase enzyme and subsequently amplified by Taq DNA polymerase. The oligonucleotide primers used in RT-PCR assay were specific to both 3' and 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 agarose gel electrophoresis. The primer pair used in present study specifically amplified VP₄ gene. The results of present study suggested that RT -PCR assay could be used for detection of rotaviruses.