Detection of murine norovirus by reverse transcription loop-mediated isothermal amplification.
Murine norovirus (MNV) has considerable genetical and biological diversity and is recognized worldwide as the most common contaminant in laboratory mouse colonies. This study developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method with the potential to detect a broad range of MNV. RT-LAMP, using a set of five primers containing mixed bases, obtained results under isothermal conditions at 62°C for 90min. Sensitivity of RT-LAMP was 50-fold less than that of two-step TaqMan real-time reverse transcription-polymerase chain reaction (TaqMan RT-PCR). Diagnostic performance of RT-LAMP on RNA extracted from mouse fecal specimens was compared with TaqMan RT-PCR and nested RT-PCR. MNV was detected in 54 of 120 mouse fecal specimens by RT-LAMP, and RT-LAMP had an estimated sensitivity and specificity of 96.4% and 100% compared with TaqMan RT-PCR, and 94.7% and 100% compared with nested RT-PCR. RT-LAMP, which does not require expensive instruments, might be useful for the screening of mice actively or persistently infected with MNV.
Department of Laboratory Animal Medicine, Institute for Biomedical Sciences, Iwate Medical University, 2-1-1 Nishi-Tokuta, Yahaba, Iwate 028-3694, Japan; Center for In Vivo Sciences, Institute for Biomedical Sciences, Iwate Medical University, 2-1-1 Nishi-Tokuta, Yahaba, Iwate 028-3694, Japan. Electronic address: hanaki@iwate-med.ac.jp.
Ken-Ichi
K
Hanaki
Experimental Animal Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
Fumio
F
Ike
Experimental Animal Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
Ayako
A
Kajita
Center for In Vivo Sciences, Institute for Biomedical Sciences, Iwate Medical University, 2-1-1 Nishi-Tokuta, Yahaba, Iwate 028-3694, Japan.
Wataru
W
Yasuno
Center for In Vivo Sciences, Institute for Biomedical Sciences, Iwate Medical University, 2-1-1 Nishi-Tokuta, Yahaba, Iwate 028-3694, Japan.
Misato
M
Yanagiba
Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
Motoki
M
Goto
Department of Virology III, National Institute of Infectious Diseases (NIID), 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
Kouji
K
Sakai
Division of Experimental Animal Research, NIID, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan.
Yasushi
Y
Ami
Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
Shigeru
S
Kyuwa
D000818:Animals; D017250:Caliciviridae Infections / Q000662:veterinary* / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000821:virology; D051379:Mice; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D048348:Reverse Transcription; D012376:Rodent Diseases / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers
Loop-mediated isothermal amplification; Murine norovirus; Nested RT-PCR; RT-LAMP; TaqMan RT-PCR