title: 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.
authors.affiliation: Gastroenteritis 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.
authors.forename: Rashi
authors.initials: R
authors.lastname: Gautam
authors.affiliation: Gastroenteritis 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.
authors.forename: Mathew D
authors.initials: MD
authors.lastname: Esona
authors.affiliation: Gastroenteritis 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.
authors.forename: Slavica
authors.initials: S
authors.lastname: Mijatovic-Rustempasic
authors.affiliation: Gastroenteritis 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.
authors.forename: Ka
authors.initials: K
authors.lastname: Ian Tam
authors.affiliation: Gastroenteritis 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.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Gentsch
authors.affiliation: Gastroenteritis 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.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Bowen
mesh_terms: D005243:Feces / Q000821:virology*; D005759:Gastroenteritis / Q000821:virology*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000821:virology; D022243:Rotavirus Vaccines / Q000008:administration & dosage / Q000009:adverse effects; D012680:Sensitivity and Specificity; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000009:adverse effects
chemical_list: C492457:RIX4414 vaccine; C492535:RotaTeq; D022243:Rotavirus Vaccines; D014613:Vaccines, Attenuated
keywords: acute gastroenteritis; qRT-PCR; rotavirus vaccine