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Updated: May 4, 2026
Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus
Published on: January 28, 2019
Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine
Rashi Gautam1, Mathew D Esona1, Slavica Mijatovic-Rustempasic1
1Gastroenteritis and Respiratory Viruses Laboratory Branch; Division of Viral Diseases; National Center for Immunization and Respiratory Diseases; Centers for Disease Control and Prevention; Atlanta, GA USA.
Insights
New quantitative real-time RT-PCR (qRT-PCR) assays can detect Rotarix and RotaTeq vaccine components in stool samples. These assays aid in monitoring vaccine strains and identifying potential vaccine-associated acute gastroenteritis (AGE).
Area of Science:
- Virology
- Vaccinology
- Molecular Diagnostics
Background:
- Group A rotaviruses (RVA) are a primary cause of severe diarrhea in young children globally.
- Live-attenuated RVA vaccines (Rotarix®, RotaTeq®) significantly reduce RVA-related mortality but can be associated with acute gastroenteritis (AGE) in vaccinees and contacts.
- Monitoring vaccine strain prevalence and detecting emergent strains in AGE cases is crucial post-vaccine introduction.
Purpose of the Study:
- To develop and validate quantitative real-time RT-PCR (qRT-PCR) assays for detecting Rotarix® and RotaTeq® vaccine components in stool samples.
- To enable high-throughput monitoring of vaccine strains in clinical and surveillance settings.
Main Methods:
- Developed specific qRT-PCR assays targeting vaccine-specific genes (Rotarix®: NSP2, VP4; RotaTeq®: VP6, VP3-WC3, VP3-human).
- Validated assays using sequence-confirmed stool samples containing vaccine strains, wild-type RVA, and RVA-negative samples.
- Generated standard curves using dsRNA transcripts for accurate quantification.
Main Results:
- Rotarix® NSP2 and VP4 assays demonstrated high sensitivity (92-100%), specificity (99-100%), and efficiency (94-105%), with limits of detection of 2-3 copies/reaction.
- RotaTeq® VP6, VP3-WC3, and VP3-human assays showed excellent sensitivity (100%), specificity (94-100%), and efficiency (91-102%), with limits of detection as low as 1-140 copies/reaction.
- The developed assays provide rapid and accurate identification of vaccine components in stool samples.
Conclusions:
- The validated qRT-PCR assays effectively detect Rotarix® and RotaTeq® vaccine components in stool.
- These assays are valuable tools for clinical and surveillance studies to monitor vaccine strain frequency.
- The assays will aid in assessing the contribution of vaccine strains to acute gastroenteritis (AGE) cases.
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.
