A broadly reactive one-step SYBR Green I real-time RT-PCR assay for rapid detection of murine norovirus.

Ken-Ichi Hanaki,Fumio Ike,Shigeru Kyuwa,Wataru Yasuno,Ayako Kajita,Kouji Sakai,Misato Yanagiba,Motoki Goto,Yasushi Ami,Shamala Devi Sekaran

doi:10.1371/journal.pone.0098108

Abstract

A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1−ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.

Highlights

Murine norovirus (MNV), a non-enveloped virus with a positive-sense single-stranded RNA genome, is a member of the genus Norovirus in the Caliciviridae family
MNV-1 causes a lethal infection with symptoms such as encephalitis, hepatitis, and pneumonia in signal transducer and activator of transcription 1 (STAT1) knockout mice, whereas MNV-O7 causes a subclinical infection in these mice [17]
101 MNV, including the sequences of 8 MNV-like viruses detected by reverse transcription-polymerase chain reaction (RT-PCR) in wild rodents in the United Kingdom and Japan, were aligned to identify highly conserved regions

Summary

Introduction

Murine norovirus (MNV), a non-enveloped virus with a positive-sense single-stranded RNA genome, is a member of the genus Norovirus in the Caliciviridae family. Thereafter, many MNV strains have been detected and/or isolated from laboratory mice and wild rodents [2,3,4,5,6]. MNV-1 causes a lethal infection with symptoms such as encephalitis, hepatitis, and pneumonia in STAT1 knockout mice, whereas MNV-O7 causes a subclinical infection in these mice [17]. MNV-1 causes a transient infection without symptoms [1,4,8] or with dose-dependent mild diarrhea in immunocompetent mice [18], whereas most MNV strains cause a long-term asymptomatic infection in immunocompetent mice with fecal viral shedding [4,16]. MNV infection does not alter the intestinal microbiota of immunocompetent mice [19]

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