title: Molecular genotyping and quantitation assay for rotavirus surveillance.
abstract: Rotavirus genotyping is useful for surveillance purposes especially in areas where rotavirus vaccination has been or will be implemented. RT-PCR based molecular methods have been applied widely, but quantitative assays targeting a broad spectrum of genotypes have not been developed. Three real time RT-PCR panels were designed to identify G1, G2, G9, G12 (panel GI), G3, G4, G8, G10 (panel GII), and P[4], P[6], P[8], P[10], P[11] (panel P), respectively. An assay targeting NSP3 was included in both G panels as an internal control. The cognate assays were also formulated as one RT-PCR-Luminex panel for simultaneous detection of all the genotypes listed above plus P[9]. The assays were evaluated with various rotavirus isolates and 89 clinical samples from Virginia, Bangladesh and Tanzania, and exhibited 95% (81/85) sensitivity compared with the conventional RT-PCR-Gel-electrophoresis method, and 100% concordance with sequencing. Real time assays identified a significantly higher rate of mixed genotypes in Bangladeshi samples than the conventional gel-electrophoresis-based RT-PCR assay (32.5% versus 12.5%, P<0.05). In these mixed infections, the relative abundance of the rotavirus types could be estimated by Cq values. These typing assays detect and discriminate a broad range of G/P types circulating in different geographic regions with high sensitivity and specificity and can be used for rotavirus surveillance.
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, United States. Electronic address: jl5yj@virginia.edu.
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, United States.
authors.forename: Kate
authors.initials: K
authors.lastname: Lurain
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.
authors.forename: Shihab U
authors.initials: SU
authors.lastname: Sobuz
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.
authors.forename: Sharmin
authors.initials: S
authors.lastname: Begum
authors.affiliation: Kilimanjaro Christian Research Institute, Moshi, Tanzania.
authors.forename: Happiness
authors.initials: H
authors.lastname: Kumburu
authors.affiliation: Kilimanjaro Christian Research Institute, Moshi, Tanzania.
authors.forename: Jean
authors.initials: J
authors.lastname: Gratz
authors.affiliation: Kilimanjaro Christian Research Institute, Moshi, Tanzania.
authors.forename: Gibson
authors.initials: G
authors.lastname: Kibiki
authors.affiliation: Division of Consolidated Laboratory Services, Virginia Department of General Services, Richmond, VA, United States.
authors.forename: Denise
authors.initials: D
authors.lastname: Toney
authors.affiliation: Division of Viral Diseases, Nation Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States.
authors.forename: Rashi
authors.initials: R
authors.lastname: Gautam
authors.affiliation: Division of Viral Diseases, Nation Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Bowen
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, United States.
authors.forename: William A
authors.initials: WA
authors.lastname: Petri
authors.affiliation: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, Bangladesh.
authors.forename: Rashidul
authors.initials: R
authors.lastname: Haque
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, United States.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
mesh_terms: D001459:Bangladesh; D002675:Child, Preschool; D060085:Coinfection / Q000453:epidemiology / Q000821:virology; D062665:Epidemiological Monitoring; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D017720:Molecular Epidemiology / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012401:Rotavirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000453:epidemiology* / Q000821:virology*; D012680:Sensitivity and Specificity; D013636:Tanzania; D019562:Viral Load / Q000379:methods*; D014768:Virginia
keywords: Diarrhea; Genotyping; Multiplex real time RT-PCR; Rotavirus