Detection of viable murine norovirus using the plaque assay and propidium-monoazide-combined real-time reverse transcription-polymerase chain reaction

摘要

Human norovirus (HuNoV) is the most common cause of gastroenteritis worldwide. The lack of a virus culture system makes it difficult to determine the viability of norovirus by only reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR). The aim of this study was to investigate the detection of viable murine norovirus (MNV) by combining propidium monoazide (PMA) or ethidium monoazide (EMA) with qRT-PCR. MNV (5.21 log(10) PFU/mL) was subjected to heat treatment at room temperature, 65, 70, 75, 80, 85, or 90 degrees C in a water bath for 1 min. The plaque assay, qRT-PCR, PMA-combined qRT-PCR, and EMA-combined qRT-PCR were then performed with heat exposed MNV samples. The MNV titer was reduced by 0.38, 1.34, and 3.71 log(10) PFU/mL at temperatures of 65, 70, and 75 degrees C, respectively. MNV was reduced >4.21 log(10) PFU/mL at 80, 85, and 90 degrees C heat inactivation. PMA (EMA) value equation for the interpretation of the viability of MNV was derived as follows: PMA (EMA) value= -log R-N - log R-p (R-N: the relative quantity value of the not-treated sample, and R-p: the relative quantity value of the PMA- or EMA-treated sample as determined by qRT-PCR). By PMA-combined qRT-PCR, the viable PMA value was 0.32, 0.83, and 2.62 for the 65, 70, and 75 degrees C preheated MNVs, respectively. The viable PMA values for the viruses heated at 80, 85, and 90 degrees C were all greater than 3.0, which was the cutoff value for discriminating between live and dead MNVs. The results of EMA-combined qRT-PCR were similar to those of qRT-PCR. Thus, PMA-combined qRT-PCR correlated well with the plaque assay in detecting viable MNVs. (C) 2015 Elsevier B.V. All rights reserved.