Abstract
Detection of viruses on berries is a challenging task, often hampered by the presence of RT-qPCR inhibiting substances from berry juice. A direct extraction method for virus detection (murine norovirus and GA phage) on frozen raspberries was previously published. We expanded (different types of berries and viruses) and improved the method using MobiSpin S400 columns that filter nucleic acids based on size-exclusion chromatography. While no inhibition was detected in filtered RNA, unfiltered RNA needed from 1:2 to more than 1:8 dilution in order to remove inhibition. The modified method gave recoveries of bovine norovirus around 40.8 ± 4.5% (40.0 ± 7.0%), 48.0 ± 26.0% (50.5 ± 7.8%), 28.3 ± 2.6% (45.8 ± 6.6%) from frozen (fresh) raspberries, strawberries and blueberries, respectively. For the same samples, recoveries of hepatitis A virus were 34.0 ± 5.9% (34.0 ± 6.0%), 40.0 ± 13.3% (34.2 ± 10.5%) and 23.0 ± 6.8% (31.5 ± 7.9%). For adenovirus40 (DNA virus), recoveries were 21.2 ± 8.6%, 16.0 ± 3.2% and 5.7 ± 0.2% from fresh raspberries, strawberries and blueberries respectively and column filtration did not add any improved effect. The modified method is effective and timesaving for detection of viral RNA from both fresh and frozen berries. As an emerging detection and direct quantification method, droplet digital RT-PCR was compared to RT-qPCR and was much less influenced by inhibitors when detecting mengovirus in unfiltered RNA from berries. However, for low levels of pure RNA, RT-qPCR showed slightly higher sensitivity and more stable results.
Highlights
During production of berries, human viral pathogens such as norovirus (NoV) and hepatitis A virus (HAV) may contaminate products through irrigation water and workers’ hands
The current method for molecular detection of virus in berries adopted by International Organization for Standardization (ISO) includes steps of elution and polyethylene glycol (PEG) precipitation, which originate from historical protocols used to recover infectious viruses from solid matrices (Lowther et al, 2019)
The procedure includes a step of pectinase digestion, as pectin is regarded as a potential room temperature (RT)-qPCR inhibitor
Summary
During production of berries (pre harvest, harvest and post harvest), human viral pathogens such as norovirus (NoV) and hepatitis A virus (HAV) may contaminate products through irrigation water and workers’ hands. The International Organization for Standardization (ISO) has adopted the PEG protocol for detection of HAV and NoV in fresh fruits and vegetables (ISO, 2017) This method is time consuming and extracted RNA may still contain RT-PCR inhibitors. A more simple and rapid direct lysis method for detecting viral genomes (murine NoV and bacteriophage GA) on frozen raspberries has been published (Perrin et al, 2015). This method was later compared to the ISO method regarding detection of human NoV in strawberries (Bartsch et al, 2016), showing comparable low recoveries (0.5 ± 0.54 versus 1.7 ± 2.3%). Droplet digital RT-PCR (RTddPCR) was compared with RT-qPCR regarding tolerance to inhibitors during detection of the viral RNA
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