ProtocolsDevelopment and application of a broadly reactive real-time reverse transcription-PCR assay for detection of murine noroviruses
Introduction
Murine norovirus (MNV) is a pathogen newly recognized in laboratory mice and a member of the genus Norovirus in the family Caliciviridae (Karst et al., 2003). The prototype strain MNV-1 was originally isolated from immunocompromised mice in the United States (Karst et al., 2003). MNV infection is currently known to occur widely among laboratory mice across the world (Goto et al., 2009, Hsu et al., 2005, Karst et al., 2003, Kelmenson et al., 2009, Kim et al., 2010, Kitajima et al., 2009, Müller et al., 2007, Mumphrey et al., 2007, Thackray et al., 2007). Therefore, MNV is now recognized as one of the most prevalent pathogens in laboratory mice (Hsu et al., 2005, Scipioni et al., 2008).
The MNV genome is a single-stranded positive-sense RNA of approximately 7.4 kb with three open reading frames (ORFs) (Karst et al., 2003). Recent studies demonstrated that the MNV genome has high genetic diversity (Müller et al., 2007, Thackray et al., 2007). The reverse transcription (RT)-polymerase chain reaction (PCR) assay is a widely used method for MNV identification (Goto et al., 2009, Hsu et al., 2006, Kim et al., 2010, Müller et al., 2007) because of its high sensitivity and applicability for further genetic analysis. A broadly reactive nested RT-PCR assay to detect MNV was recently developed, and MNVs were identified in laboratory mice in Japan (Kitajima et al., 2009).
The real-time RT-PCR assay is a rapid method for detecting viral RNA with less cross-contamination, thereby allowing quantitative analysis. Several TaqMan-based real-time RT-PCR assays targeting ORF1 (Belliot et al., 2008), the ORF1–ORF2 junction region (Bae and Schwab, 2008, Baert et al., 2008, Stals et al., 2009), and ORF2 region (Hewitt et al., 2009, Lee et al., 2008, Müller et al., 2007) have been reported recently. However, primer sets and probes used in those studies were designed based on the MNV-1 strain sequence (accession no. AY228235), and most of these primer sets and probes were found to have a considerable number of mismatches with other MNV strains when full- or nearly full-length genome nucleotide sequences of 44 MNV strains were aligned (data not shown).
In the present study, a TaqMan-based real-time RT-PCR assay for quantitative detection of a broad range of genetically diverse MNV strains was developed and applied for quantitation of MNV-RNA in murine stool specimens and in the supernatant during in vitro replication.
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Cells, virus and infectivity assay
A cell line derived from mouse leukemic monocyte macrophages, RAW 264.7 (ATCC TIB-71), was grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. An MNV strain, S7-PP3, was isolated from a murine fecal specimen in Japan and purified by three successive plaque clonings in RAW 264.7 cells (Tohya et al., unpublished).
Infectivity of MNV was measured by an endpoint titration assay using a 96-well microtiter plate. Subconfluent
Development of quantitative real-time RT-PCR
To design broadly reactive primers and a TaqMan MGB probe, the nucleotide sequences of the 44 MNV strains were aligned, and among which a part (129 nts) located on the 3′-terminus of ORF1 and the 5′-terminus of ORF2 was selected as the target region of the real-time RT-PCR assay (Fig. 1). A real-time RT-PCR assay, utilizing a forward primer (MNV-S primer), a reverse primer (MNV-AS primer), and a TaqMan MGB probe (MNV-TP) was developed (Table 1 and Fig. 1). A nucleotide BLAST search of sequences
Discussion
In the present study, a quantitative real-time RT-PCR assay utilizing newly designed primers and a TaqMan MGB probe was developed to detect a broad range of genetically diverse MNV strains. Highly conserved sequences at the ORF1–ORF2 junction region among MNV strains were found in a previous study (Kitajima et al., 2009), and the primer set and a TaqMan MGB probe for the real-time RT-PCR assay were designed (Fig. 1).
Although several real-time RT-PCR assays have been developed for MNVs (Bae and
Acknowledgments
This study was supported in part by grants from the Ministry of Health, Labour, and Welfare of Japan and the Japan Health Science Foundation.
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2021, Environmental ResearchCitation Excerpt :MNV was spiked as surrogate because it is a positive-sense single-stranded RNA virus like SARS-CoV-2, and will likely be similar in terms of RNA extraction efficiency. Quantitative PCR (qPCR) for MNV was performed using forward primer (5′- CCGCAGGAACGCTCAGCAG-3′), reverse primer (5′- GGYTGAATGGGGACGGCCTG-3′) and Taqman probe (5′- FAM-ATGAGTGATGGCGCA-ZEN/IBFQ-3′) (Integrated DNA Technologies, Leuven, Belgium (Kitajima et al., 2010). A six-points standard curve was generated using a synthetic oligonucleotide (gblocks@ gene fragment, Integrated DNA technologies, IA, USA) containing 300–350 bp DNA sequence that encompasses a complementary region for which the primers and probes would anneal to (Table S1).