Purpose: The aim of this work was to determine the suitability of using murine norovirus (MNV-1) as an extraction process control for the improved molecular detection of enteric viruses in fresh strawberries and raspberries and frozen raspberries by real-time RT-PCR.
Methods: Fresh strawberries and raspberries or frozen raspberries from local grocery stores were washed with 10% trisodium phosphate, allowed to air-dry under ultraviolet light and aseptically surface-spiked with 5 log PFU/ml MNV-1 (as an extraction process control) or with phosphate buffered saline (PBS, negative control). Viruses were first eluted with PBS containing pectinase, followed with TRIzol™ for RNA extraction, and passed through a QIAshredder. Ten-fold diluted RNA extracts from MNV-1 stock, and spiked and control berries were used for detection by SYBR green I real-time RT-PCR. 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 could detect MNV-1 up to -7 log diluted RNA (corresponding to <1 PFU/ml), indicating 7 log RT-PCR units in the stock. Fresh strawberries and raspberries spiked with MNV-1 showed detection to -6 log diluted RNA extracts (corresponding detection up to 1 log RT-PCR units) by both RT-PCR and gel electrophoresis. Frozen raspberries showed detection to -8 log dilution (corresponding to < 0.1 PFU/ml or -1 log RT-PCR units) by both RT-PCR and gel electrophoresis.
Significance: The simple pectinase-TRIzol extraction method showed that MNV-1 was a suitable extraction process control to rapidly detect human enteric viruses from berries by RT-PCR to prevent berry-related outbreaks worldwide.