Murine Norovirus (MNV-1) Detection in Low-water Activity Foods by Real-time Reverse Transcription-Polymerase Chain Reaction

Introduction: In Mexico, there is a lack of information regarding the incidence of pathogens in low water activity (a_w) food items. Most of these products are sold in bulk, and thus follow-up safety procedures during outbreaks become complicated. Human norovirus surveillance and studies on their ability to survive in low a_w foods and drying conditions applied in food industry are needed to determine transmission risk.

Purpose: The aim of this project was to rapidly detect murine norovirus (MNV-1, as an extraction control and surrogate for human noroviruses) from low a_w foods using real-time RT-PCR.

Methods: Individual 25-g samples of peanuts, pecans, raisins, and sun-dried tomatoes purchased from local supermarkets were kept under ultraviolet light for 10 min, and aseptically surface-spiked with 5 log PFU/ml MNV-1 (positive extraction control). Viruses were eluted using TRIzol^TM, RNA extracted and passed through a QIAshredder. SYBR green I-based RT-PCR was carried out on ten-fold diluted RNA extracts from MNV-1 stock and spiked samples. Agarose gel electrophoresis and Tm analysis were used to confirm product size. All experiments were replicated thrice.

Results: TRIzol RNA extraction followed by RT-PCR was able to detect MNV-1 stock up to -7 log RNA dilution (1 PFU/ml). Peanuts and pecans spiked with MNV-1 showed detection to -3 log dilution (~4 log PFU/ml or 4 log RT-PCR units) and to -5 log dilution (~2 log PFU/ml or 2 log RT-PCR units), respectively. Sun-dried tomatoes showed detection up to ~10 log PFU/ml (1 log RT-PCR units) and raisins up to 1 log PFU/ml.

Significance: TRIzol RNA extraction with RT-PCR detection showed that MNV-1 could be used as an extraction process control for human enteric virus detection from low a_w foods, though needs further optimization for improved detection.