title: The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting rotavirus A and norovirus.
abstract: Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits.
authors.affiliation: The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.
authors.forename: Tran Thi Ngoc
authors.initials: TT
authors.lastname: Dung
authors.forename: Voong Vinh
authors.initials: VV
authors.lastname: Phat
authors.forename: Tran Vu Thieu
authors.initials: TV
authors.lastname: Nga
authors.forename: Phan Vu Tra
authors.initials: PV
authors.lastname: My
authors.forename: Pham Thanh
authors.initials: PT
authors.lastname: Duy
authors.forename: James I
authors.initials: JI
authors.lastname: Campbell
authors.forename: Cao Thu
authors.initials: CT
authors.lastname: Thuy
authors.forename: Nguyen Van Minh
authors.initials: NV
authors.lastname: Hoang
authors.forename: Pham
authors.initials: P
authors.lastname: Van Minh
authors.forename: Hoang
authors.initials: H
authors.lastname: Le Phuc
authors.forename: Pham Thi Ngoc
authors.initials: PT
authors.lastname: Tuyet
authors.forename: Ha
authors.initials: H
authors.lastname: Vinh
authors.forename: Duong Thi Hue
authors.initials: DT
authors.lastname: Kien
authors.forename: Huynh Le Anh
authors.initials: Hle A
authors.lastname: Huy
authors.forename: Nguyen Thanh
authors.initials: NT
authors.lastname: Vinh
authors.forename: Tran Thi Thu
authors.initials: TT
authors.lastname: Nga
authors.forename: Nguyen Thi Thu
authors.initials: NT
authors.lastname: Hau
authors.forename: Nguyen Tran
authors.initials: NT
authors.lastname: Chinh
authors.forename: Tang Chi
authors.initials: TC
authors.lastname: Thuong
authors.forename: Ha Manh
authors.initials: HM
authors.lastname: Tuan
authors.forename: Cameron
authors.initials: C
authors.lastname: Simmons
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Farrar
authors.forename: Stephen
authors.initials: S
authors.lastname: Baker
mesh_terms: D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology; D002675:Child, Preschool; D003967:Diarrhea / Q000175:diagnosis / Q000821:virology; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis / Q000235:genetics; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D060885:Multiplex Polymerase Chain Reaction; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000821:virology
chemical_list: D012367:RNA, Viral