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

https://doi.org/10.1016/j.jviromet.2014.03.025Get rights and content

Highlights

  • RT-LAMP method with the potential to detect a broad range of MNV was developed.

  • Sensitivity of RT-LAMP was 50-fold less than that of TaqMan RT-PCR.

  • In a practical diagnostic test, RT-LAMP exhibited 96.4% and 94.7% sensitivity compared with TaqMan RT-PCR and nested RT-PCR.

  • In a practical diagnostic test, RT-LAMP exhibited 100% specificity compared with both TaqMan RT-PCR and nested RT-PCR.

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 90 min. 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

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