One-step quantitative RT-PCR for the detection of rotavirus in acute gastroenteritis.

The standard diagnosis of rotavirus gastroenteritis is based on the demonstration of rotavirus antigen in stools using an enzyme immunoassay (EIA). In this study, a one-step quantitative RT-PCR (Q-PCR) was used for sensitive detection of rotavirus in diarrheal stools. The primers and TaqMan probe for the Q-PCR were selected from a highly conserved region of the non-structural protein 3 (NSP3) of rotavirus. After validation, the test was applied to study rotavirus EIA positive (N=25) and EIA negative (N=143) stool specimens from cases of acute gastroenteritis of all degrees of severity in a prospective follow-up cohort of infants from 2 months to 2 years of age. Q-PCR detected all 25 EIA positive rotavirus antigens and seven additional cases that were rotavirus EIA negative, i.e. 28% more rotavirus positive cases than identified by EIA. It is concluded that Q-PCR using primers targeted at NSP3 is a rapid and sensitive method for diagnosing acute rotavirus gastroenteritis.