Rapid concentration and detection of hepatitis A virus from lettuce and strawberries.
Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex 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 hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece 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 IM-PCR. However, considering the large volumes (< or =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 < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.
Bidawid
S
S
Food Directorate, Bureau of Microbial Hazards, Sir F.G. Banting Research Centre, Ross Avenue, Tunney's Pasture, P.O. Box 2204A2, Ottawa, Ont., Canada K1A OL2. sabah_bidawid@hc-sc.gc.ca
Farber
J M
JM
Sattar
S A
SA
D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D005638:Fruit / Q000821:virology*; D006507:Hepatovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D018189:Immunomagnetic Separation / Q000379:methods; D018545:Lactuca / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D012367:RNA, Viral