Rapid concentration and detection of hepatitis A virus from lettuce and strawberries

Immunomagnetic brads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentr ate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and st rawberries experimentally contaminated. Direct reverse transcriptase-polyme rase chain reaction IRT-PCR) amplification of the collected HAV-beads compl ex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundre d-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the f ilters and the captured virus was eluted with 10 ml volumes of 1% beef extr act. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 mu l from this eluate failed to pro duce a clear positive band signal. As little as 10 PFU, present on each pie ce of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the I M-PCR. However, considering the large volumes ( less than or equal to 50 ml ) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to less than or equal to 20 m l volumes. These data indicate that the F-IM-PCR method provides the potent ial for a greater sensitivity of detection than the IM-PCR, since low level s of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater a mount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplif ication from beef extract eluates was not successful in detecting HAV from produce. (C) 2000 Elsevier Science B.V. All rights reserved.