Sensitive and Specific Quantitative Detection of Rotavirus A by One- Step Real-Time Reverse Transcription-PCR Assay without Antecedent

A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophi-lus (rTth) enzyme was developed to detect and quantify rotavirus A (RVA). By using rTth polymerase, significant improvement was achieved over the existing real-time RT-PCR assays, which require denaturation of the RVA double-stranded RNA (dsRNA) prior to assay setup. Using a dsRNA transcript for segment 7, which encodes the assay target NSP3 gene, the limit of detection for the improved assay was calculated to be approximately 1 genome copy per reaction. The NSP3 qRT-PCR assay was validated us-ing a panel of 1,906 stool samples, 23 reference RVA strains, and 14 nontarget enteric virus samples. The assay detected a diverse number of RVA genotypes and did not detect other enteric viruses, demonstrating analytical sensitivity and specificity for RVA in testing stool samples. A XenoRNA internal process control was introduced and detected in a multiplexed qRT-PCR format. Because it does not require an antecedent dsRNA denaturation step, this assay reduces the possibility of sample cross-contami-nation and requires less hands-on time than other published qRT-PCR protocols for RVA detection. Group A rotaviruses (RVA) are estimated to cause 453,000deaths among infants and young children each year (1). The genome of RVA, members of the Reoviridae family, consists of 11 double-stranded RNA (dsRNA) segments which encode six viral structural proteins (VP1 to VP4, VP6, and VP7) and six nonstruc-tural proteins (NSP1 to NSP6) (2). Surrounding the dsRNA ar