Detection of murine norovirus by reverse transcription loop-mediated isothermal amplification

Ken-Ichi Hanaki; Fumio Ike; Ayako Kajita; Wataru Yasuno; Misato Yanagiba; Motoki Goto; Kouji Sakai; Yasushi Ami; Shigeru Kyuwa

doi:10.1016/j.jviromet.2014.03.025

Detection of murine norovirus by reverse transcription loop-mediated isothermal amplification

J Virol Methods. 2014 Aug:204:17-24. doi: 10.1016/j.jviromet.2014.03.025. Epub 2014 Apr 6.

Authors

Ken-Ichi Hanaki¹, Fumio Ike², Ayako Kajita², Wataru Yasuno³, Misato Yanagiba³, Motoki Goto⁴, Kouji Sakai⁵, Yasushi Ami⁶, Shigeru Kyuwa⁷

Affiliations

¹ 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.
² Experimental Animal Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
³ Center for In Vivo Sciences, Institute for Biomedical Sciences, Iwate Medical University, 2-1-1 Nishi-Tokuta, Yahaba, Iwate 028-3694, Japan.
⁴ Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
⁵ Department of Virology III, National Institute of Infectious Diseases (NIID), 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.
⁶ Division of Experimental Animal Research, NIID, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan.
⁷ Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.

Abstract

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.

Keywords: Loop-mediated isothermal amplification; Murine norovirus; Nested RT-PCR; RT-LAMP; TaqMan RT-PCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Caliciviridae Infections / veterinary*
Caliciviridae Infections / virology
DNA Primers / genetics
Feces / virology
Mice
Norovirus / genetics
Norovirus / isolation & purification*
Nucleic Acid Amplification Techniques / methods*
Reverse Transcription*
Rodent Diseases / diagnosis*
Rodent Diseases / virology*
Sensitivity and Specificity
Temperature
Time Factors

Substances

DNA Primers