Development and application of a broadly reactive real-time reverse transcription-PCR assay for detection of murine noroviruses.
Murine norovirus (MNV) is a viral agent newly identified in laboratory mice and a large number of genetically diverse MNV strains have been reported to date. A broadly reactive TaqMan-based real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay was developed for MNVs. Novel primers and a TaqMan MGB probe were designed targeting highly conserved sequences among MNV strains, which are located in the open reading frames 1 (ORF1)-ORF2 junction region. The quantitative range of this assay was determined as 1.0 × 10(2)-1.0 × 10(8)copies/PCR tube based on a 10-fold serial dilution of plasmid DNA containing the target sequences. Viral RNA in eight murine stool specimens positive by nested RT-PCR assay was measured, and the highest viral RNA load was calculated at 4.7 × 10(6)copies/g-stool. MNV was inoculated into RAW 264.7 cells, and the viral RNA was monitored to validate assay sensitivity. MNV-RNA in the supernatant was detected during in vitro replication, which increased substantially from 5 to 30 h post-infection (hpi) and reached more than 1.0 × 10(10)copies/mL at 96 hpi. This real-time RT-PCR assay is a useful tool to detect and quantify MNV-RNA in in vivo and in vitro studies.
Kitajima
Masaaki
M
Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. kitajima@env.t.u-tokyo.ac.jp
Oka
Tomoichiro
T
Takagi
Hirotaka
H
Tohya
Yukinobu
Y
Katayama
Hiroyuki
H
Takeda
Naokazu
N
Katayama
Kazuhiko
K
D000818:Animals; D017250:Caliciviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000821:virology; D008264:Macrophages / Q000821:virology; D051379:Mice; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012376:Rodent Diseases / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity; D019562:Viral Load; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D012367:RNA, Viral