title: Pitfalls in mouse norovirus (MNV) detection in fecal samples using RT-PCR, and construction of new MNV-specific primers.
abstract: The murine norovirus (MNV), which belongs to the Caliciviridae family, is prevalent in laboratory mice. Since this virus affects macrophages and dendritic cells, infected mice are not suitable for immunological investigations, making it important to detect MNV infections accurately. When we tested RNA extracts derived from mouse feces for MNV detection using nested RT-PCR with a set of MNV-specific primers reported by Goto et al. (Exp. Anim. 58: 135-140, 2009), we found that these primers amplified not only an MNV-specific signal but also amplified a relatively weak signal with a size almost identical to that of the specific signal. Analysis of the nucleotide sequence of this amplified signal revealed that it was at least 98% identical to the exophosphatase gene of a commensal bacterium, Bacteroides vulgatus. Subsequent analysis showed that the signal amplified with a pair of nested primers was from DNA derived from B. vulgatus, which is sometimes present in SPF laboratory mouse feces, and the nested primers used were both partly homologous with the B. vulgatus nucleotide sequence. We thus designed a new set of nested RT-PCR primers that was not cross-reactive with the B. vulgatus genome. PCR products amplified by the newly designed primers were at least 89.3% identical to the MNV RNA polymerase gene in all cases. Our findings demonstrated that the primer set we designed was suitable for detecting an MNV-specific signal without cross-reacting with B. vulgatus DNA in mouse feces.
authors.affiliation: The Institute of Experimental Animal Sciences, Faculty of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tajima
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kotani
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kurosawa
authors.forename: Masayuki
authors.initials: M
authors.lastname: Miyasaka
mesh_terms: D000818:Animals; D001439:Bacteroides / Q000201:enzymology / Q000235:genetics; D017250:Caliciviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology*; D017931:DNA Primers; D004269:DNA, Bacterial; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005243:Feces / Q000821:virology*; D005260:Female; D005759:Gastroenteritis / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology*; D051379:Mice; D008813:Mice, Inbred ICR; D029322:Norovirus / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012376:Rodent Diseases / Q000175:diagnosis* / Q000821:virology*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases