title: Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR.
abstract: We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.
authors.affiliation: Section of Infectious Disease, R&D Center, BML, Kawagoe, Saitama 350-1101, Japan. tkage@alk.co.jp
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kageyama
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kojima
authors.forename: Michiyo
authors.initials: M
authors.lastname: Shinohara
authors.forename: Kazue
authors.initials: K
authors.lastname: Uchida
authors.forename: Shuetsu
authors.initials: S
authors.lastname: Fukushi
authors.forename: Fuminori B
authors.initials: FB
authors.lastname: Hoshino
authors.forename: Naokazu
authors.initials: N
authors.lastname: Takeda
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Katayama
mesh_terms: D001483:Base Sequence; D017250:Caliciviridae Infections / Q000453:epidemiology / Q000821:virology; D017931:DNA Primers; D004196:Disease Outbreaks; D005759:Gastroenteritis / Q000453:epidemiology / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D029322:Norovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers