Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus

Nucleic acid amplification techniques are used frequently for rapid diagnosis of viral diseases. In this study, a real-time polymerase chain reaction protocol that uses primers specific for the viral VP4 gene and the commercial SYBR Green reagent were evaluated for the quantitative measurement of human rotavirus (HRV) RNA in human stool specimens. SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. The assay resulted in a sensitive and reproducible detection of targets ranging from low (<10(2)rotavirus cDNA copies/reaction) to high numbers (>10(6)rotavirus cDNA copies/reaction). No cross-reaction was found with crude cell culture stocks of coxsackievirus, echovirus, poliovirus, hepatitis A virus and adenovirus. Analysis with the HRV cDNA standard demonstrated high reproducibility with a coefficient of variation (CV) of 0.2-0.9%. Daily performance among three different laboratories showed a CV no greater than 8%, indicating an intermediate level of variation. These results demonstrate the feasibility of this method for quantitative analysis of human rotavirus in clinical samples.