Development of real-time RT-PCR assay for diagnosis of viral enteritis in neonatal goat kids

Abstract

Rotavirus gastroenteritis is a worldwide disease affecting primarily infants, young children and young ones of wide variety of mammalian and avian species. Diarrhoea in goat kids is most frequently found associated with Group A rotavirus (GARV) and another enteric pathogen bovine coronavirus (BCoV), a major viral pathogen associated with neonatal diarrhoea. Enteric BCoV replicates in epithelial cells of gut, destroying villi, resulting in severe, often bloody diarrhoea in calves. It requires highly sensitive and specific assays to diagnose the disease at field level. In the present study, a real-time reverse-transcriptase (RT) polymerase chain reaction (PCR) were developed and validated for specific detection and quantification of GARV and BCoV with high sensitivity and specificity. For real-time RT-PCR, primers were designed to target nucleocapsid gene for BCoV; NSP4 gene and VP6 gene were designed for GARV using discontiguous conserved sequences. Real-time RT-PCR assay was standardized by serial dilution of positive GARV and BCoV RNA. The rotavirus real-time RT-PCR assay was found to be specific to rotavirus, but broadly reactive to GARV. The sensitivity of the assay for detecting rotavirus and BCoV in faecal samples and tissue sample was found to be high in such reactions. The real-time RT-PCR assay was effective in detecting GARV and BCoV in all positive samples obtained from sheds, farms and outbreaks. The results of this study demonstrate that the real-time RT-PCR assay for viral enteritis is broadly reactive, specific, and sensitive for detection of GARV and BCoV in faecal sample and tissue samples.