Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix® and RotaTeq® are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix® and RotaTeq® vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix® and RotaTeq® vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix® (NSP2, VP4) and RotaTeq® (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix® NSP2 and VP4 qRT-PCR assays exhibited 92–100% sensitivity, 99–100% specificity, 94–105% efficiency, and a limit of detection of 2–3 copies per reaction. RotaTeq® VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94–100% specificity, 91–102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix® and RotaTeq® vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE.

A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophilus (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 using 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-contamination and requires less hands-on time than other published qRT-PCR protocols for RVA detection. Group A rotaviruses (RVA) are estimated to cause 453,000 deaths 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 nonstructural proteins (NSP1 to NSP6) (2). Surrounding the dsRNA are three concentric protein layers, namely, a central core (VP2), a middle protein layer (VP6), and an outer capsid composed of VP7 and VP4 proteins, while the viral RNA-dependent RNA polymerase (VP1) and the RNA capping enzyme (VP3) are packaged within the core shell (3). Traditionally, viral classification has been based on the serological characteristics and sequence diversity of the outer capsid proteins, VP7 (glycosylated, G-type) and VP4 (protease sensitive, P-type) (4). Although 27 G genotypes and 35 P genotypes have been identified to date (5) Numerous techniques have been developed to detect RVA in stool samples. These techniques include, but are not limited to, virus isolation in cell culture, electron microscopy (EM), enzyme immunoassays (EIA), coupled reverse transcription and PCR amplification (RT-PCR), and real-time quantitative RT-PCRs (qRTPCRs) (2, 7-19). The molecular techniques are more rapid than the cell culture-based techniques and are more sensitive than EM or EIA. The threshold for detection of RVA by EM is approximately 10 7 viral particles/ml of stool (20). EIAs are 10 to 100 times more sensitive than EM assays, but the sensitivity and specificity of EIAs are more variable (21-23). A number of studies using RT-PCR assays targeting the RVA VP4, VP6, and VP7 gene segments have shown increases of 15% to 27% in the rate of RVA detection in comparison with . RT-PCR assays employ either one-step or two-step reverse transcription followed by an ...