title: Evaluation of Various Real-Time Reverse Transcription Quantitative PCR Assays for Norovirus Detection.
abstract: Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.
authors.affiliation: Department of Environmental Health Sciences, Graduate School of Public Health, Seoul National University, Seoul 08826, Republic of Korea.
authors.forename: Ju Eun
authors.initials: JE
authors.lastname: Yoo
authors.affiliation: Department of Environmental Health Sciences, Graduate School of Public Health, Seoul National University, Seoul 08826, Republic of Korea.|Institute of Health and Environment, Seoul National University, Seoul 08826, Republic of Korea.
authors.forename: Cheonghoon
authors.initials: C
authors.lastname: Lee
authors.affiliation: Department of Environmental Health Sciences, Graduate School of Public Health, Seoul National University, Seoul 08826, Republic of Korea.|N-Bio, Seoul National University, Seoul 08826, Republic of Korea.|KoBioLabs, Inc., Seoul 08826, Republic of Korea.
authors.forename: SungJun
authors.initials: S
authors.lastname: Park
authors.affiliation: Department of Environmental Health Sciences, Graduate School of Public Health, Seoul National University, Seoul 08826, Republic of Korea.|N-Bio, Seoul National University, Seoul 08826, Republic of Korea.|KoBioLabs, Inc., Seoul 08826, Republic of Korea.|Center for Human and Environmental Microbiome, Seoul National University, Seoul 08826, Republic of Korea.
authors.forename: GwangPyo
authors.initials: G
authors.lastname: Ko
mesh_terms: D001483:Base Sequence; D017250:Caliciviridae Infections / Q000175:diagnosis / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004196:Disease Outbreaks; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis / Q000821:virology; D006801:Humans; D057230:Limit of Detection; D029322:Norovirus / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D012367:RNA, Viral / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: Norovirus; RT-qPCR; detection limit; internal quencher; sensitivity