Detection of Viable Murine Norovirus Using Plaque Assay and Propidium Monoazide Real-time Reverse Transcription-polymerase Chain Reaction

Introduction: Norovirus is the most common cause of gastroenteritis worldwide. Lacking of virus culture system, it is hard to determine viability of norovirus only by reverse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR.

Purpose: The aim of this study was to investigate the detection of viable murine norovirus (MNV) by the combination of propidium monoazide (PMA) treatment and real-time RT-PCR.

Methods: Five log PFU/ml MNV-1 was inactivated by the heat treatment at 20°C, 60°C, 70°C, 80°C, and 100°C in water bath for 1 min. Plaque assay, real-time RT-PCR, and PMA-combined real-time RT-PCR was performed with heat-treated MNV.

Results: The titer of MNV was reduced 0.52, 1.00, and 2.00 log PFU/ml at heat-inactivated MNV at 60°C, 70°C, and 80°C condition, respectively. MNV were not detected at 100°C condition by plaque assay. The relative quantity values of MNV reduced 0.10 log, 0.88 log, 1.09 log, and 1.55 log at 60°C, 70°C, 80°C, and 100°C condition, respectively, by real-time RT-PCR. However, in PMA-combined real-time RT-PCR group, the relative quantity values reduced 0.47 log, 1.93 log, 2.80 log, and 3.89 log at 60°C, 70°C, 80°C, and 100°C condition, respectively.

Significance: PMA-combined real-time RT-PCR was correlated with plaque assay to detect viable MNV.