title: Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.
abstract: Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (103DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.
authors.affiliation: Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Kitwadee
authors.initials: K
authors.lastname: Rupprom
authors.affiliation: Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Porntip
authors.initials: P
authors.lastname: Chavalitshewinkoon-Petmitr
authors.affiliation: Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Pornphan
authors.initials: P
authors.lastname: Diraphat
authors.affiliation: Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, 10400, Thailand. leera.kit@mahidol.ac.th.
authors.forename: Leera
authors.initials: L
authors.lastname: Kittigul
mesh_terms: D017250:Caliciviridae Infections / Q000175:diagnosis*; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: genogroup; norovirus; quantification; real-time RT-PCR