Broadly reactive TaqMan^® assay for real-time RT-PCR detection of rotavirus in clinical and environmental samples

Abstract

Rotaviruses are enteric pathogens responsible for a significant burden of disease, especially in children, through person-to-person transmission and exposure to contaminated food and water. In the present study, a TaqMan^® probe-based real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and validated for sensitive and specific detection and quantification of rotavirus for the routine screening of clinical and environmental samples. The assay primers and probes were designed to target the non-structural protein region 3 (NSP3) of rotavirus. The rotavirus real-time RT-PCR assay was found to be specific to rotavirus, but broadly reactive to rotavirus genogroups 1-4, 9, 10 and 12. Specificity testing did not identify any cross-reactivity of the assay with a panel of 36 non-rotavirus enteric virus specimens. The sensitivity of the assay was determined using quantified rotavirus stocks and a plasmid DNA stock. Estimated detection limits in reagent-grade water were five genome equivalent copies (GEC) per reaction and two to four rotavirus particles per reaction. The sensitivity of the assay for detecting rotaviruses in environmental water samples was found to be six virus particles per reaction. The rotavirus real-time RT-PCR assay was effective in detecting rotavirus in all 79 stool specimens obtained from a hospital in India. The results of this study demonstrate that the real-time RT-PCR assay for rotavirus is broadly reactive, specific, and sensitive for detection of rotaviruses in clinical specimens and water samples.