Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine strains in stool samples

Abstract

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.

Keywords: acute gastroenteritis; qRT-PCR; rotavirus vaccine.

Figure 1. Alignment showing Rotarix^® and RotaTeq^® qRT-PCR Assay Target Regions. Each primer and probe target region is shown aligned with the corresponding region of non-target strains. Numbers are nucleotide coordinates refer to positions in sequence listed at the top of each group. Bold text indicates nucleotide substitutions incorporated into primer and probes sequences to provide assay specificity. A dash indicates a base identical to the aligned residue in the reference strain.

Figure 2. Performance of the Rotarix^® NSP2 qRT-PCR Assay. (A) Amplification plot of Rotarix^® NSP2 qRT-PCR assay using sequence confirmed Rotarix^® positive stool samples. (B) Amplification plot of Rotarix^® NSP2 qRT-PCR assay using sequence confirmed wild-type stool samples. (C) Amplification plot of Rotarix^® NSP2 qRT-PCR assay using 10-fold serial dilutions of Rotarix^® NSP2 vaccine derived dsRNA transcript. (D) Correlation between Rotarix^® NSP2 qRT-PCR assay threshold value (Ct) and log copy number.

Figure 3. Performance of the RotaTeq^® VP6 qRT-PCR Assay. (A) Amplification plot of RotaTeq^® VP6 qRT-PCR assay using sequence confirmed RotaTeq^® positive stool samples. (B) Amplification plot of RotaTeq^® VP6 qRT-PCR assay using sequence confirmed wild-type stool samples. (C) Amplification plot of RotaTeq^® VP6 qRT-PCR assay using 10-fold serial dilutions of RotaTeq^® VP6 vaccine derived dsRNA transcript. (D) Correlation between RotaTeq^® VP6 qRT-PCR assay threshold value (Ct) and log copy number.