Bidawid S, Farber J M, Sattar S A
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.
J Virol Methods. 2000 Aug;88(2):175-85. doi: 10.1016/s0166-0934(00)00186-5.
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.
免疫磁珠 - 聚合酶链反应（IM - PCR）、带正电荷的病毒吸附过滤器（F）或两种方法的组合（F - IM - PCR）被用于在实验污染的生菜和草莓样本中捕获、浓缩并快速检测甲型肝炎病毒（HAV）。对收集到的HAV - 磁珠复合物进行直接逆转录 - 聚合酶链反应（RT - PCR）扩增显示，在1毫升来自农产品的洗涤液中存在的病毒的检测限为0.5个空斑形成单位（PFU），这比RT - PCR显示的灵敏度高数百倍。在单独的试验中，将来自农产品的含病毒洗涤液通过过滤器，并用10毫升体积的1%牛肉提取物洗脱捕获的病毒。在过滤器捕获的62%的HAV中，平均有34.8%被1%牛肉提取物洗脱。对该洗脱液的2微升进行PCR扩增未能产生清晰的阳性条带信号。F - IM - PCR可检测到每片生菜或草莓上低至10 PFU的病毒，其灵敏度几乎比IM - PCR的0.5 PFU检测限低20倍。然而，考虑到F - IM - PCR中使用的大量体积（≤50毫升），其检测灵敏度可能远高于IM - PCR，后者限于≤20毫升的体积。这些数据表明，F - IM - PCR方法比IM - PCR具有更高的检测灵敏度潜力，因为与IM - PCR方法相比，它能从大量样本中检测到低水平的病毒。尽管带正电荷的过滤器捕获的病毒量比IM - PCR和F - IM - PCR方法都多，但从牛肉提取物洗脱液中直接进行PCR扩增未能成功检测到农产品中的HAV。
