Extended direct lysis method for virus detection on berries including droplet digital RT-PCR or real time RT-PCR with reduced influence from inhibitors

摘要

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 adeno-virus40 (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.