A broadly reactive one-step SYBR Green I real-time RT-PCR assay for rapid detection of murine norovirus.
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.
Department of Laboratory Animal Medicine, Institute for Biomedical Sciences, Iwate Medical University, Yahaba, Iwate, Japan; Center for In Vivo Sciences, Institute for Biomedical Sciences, Iwate Medical University, Yahaba, Iwate, Japan.
Ken-Ichi
K
Hanaki
Experimental Animal Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
Fumio
F
Ike
Experimental Animal Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
Ayako
A
Kajita
Center for In Vivo Sciences, Institute for Biomedical Sciences, Iwate Medical University, Yahaba, Iwate, Japan.
Wataru
W
Yasuno
Center for In Vivo Sciences, Institute for Biomedical Sciences, Iwate Medical University, Yahaba, Iwate, Japan.
Misato
M
Yanagiba
Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Bunkyo, Tokyo, Japan.
Motoki
M
Goto
Department of Virology III, National Institute of Infectious Diseases (NIID), Musashimurayama, Tokyo, Japan.
Kouji
K
Sakai
Division of Experimental Animal Research, NIID, Shinjuku, Tokyo, Japan.
Yasushi
Y
Ami
Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo, Japan.
Shigeru
S
Kyuwa
D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D002460:Cell Line; D017931:DNA Primers; D003959:Diamines; D005243:Feces / Q000821:virology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D016366:Open Reading Frames; D009930:Organic Chemicals / Q000737:chemistry*; D010957:Plasmids; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I