U.S. Food and Drug Administration, Division of Seafood Science and Technology, Gulf Coast Seafood Laboratory, Dauphin Island, Alabama 36528, USA.
J Food Prot. 2009 Oct;72(10):2106-9. doi: 10.4315/0362-028x-72.10.2106.
The applicability of real-time PCR was examined for detection of vibrios from postharvest-processed (PHP) oysters to allow for a more rapid assay and higher sample throughput than currently used. During June to October 2004, 68 PHP oyster samples were collected directly from PHP firms or from retail markets across the United States. PHP oysters were examined to determine the effectiveness of treatments in the reduction of vibrio levels and to compare the analytical methods utilized. The latter is the focus of the data presented here. Each sample was analyzed for Vibrio parahaemolyticus and V. vulnificus by using a 2-dilution, three-tube most-probable-number (MPN) and a 25-g presence/absence enrichment in alkaline peptone water. Following 6-h and overnight enrichment, aliquots from each MPN tube and the 25-g sample were streaked onto selective media and tested by real-time PCR. Colonies from the selective agar were confirmed as V. parahaemolyticus or V. vulnificus by DNA colony hybridization. DNA hybridization and real-time PCR results for each MPN tube and the 25-g enrichment at both time points were analyzed individually for each organism. The methods were in agreement for 857 (95%) of 901 and for 882 (98%) of 903 tubes for detection of V. parahaemolyticus and V. vulnificus, respectively. Overall, there was 96% agreement between real-time and DNA colony hybridization. The results obtained by real-time PCR were comparable to those from DNA colony hybridization, but analysis time was significantly reduced for the detection of vibrios in PHP-treated oysters.
实时 PCR 法检测经加工的牡蛎中的弧菌，与目前所用方法相比，该法可提高检测速度和样本通量。2004 年 6 月至 10 月，从美国各地的加工企业或零售市场直接采集了 68 份经加工的牡蛎样本。检测经加工的牡蛎，以确定处理方法在降低弧菌水平方面的效果，并比较所使用的分析方法。本文重点介绍后者。采用两稀释度、三管最可能数（MPN）和 25g 碱性蛋白胨水存在/不存在增菌法，每份样本均检测副溶血性弧菌和创伤弧菌。6h 和过夜增菌后，从每个 MPN 管和 25g 样品中取等分试样，划线接种于选择性培养基，并用实时 PCR 进行检测。选择性琼脂上的菌落通过 DNA 菌落杂交确认为副溶血性弧菌或创伤弧菌。单独分析每个 MPN 管和 25g 增菌在两个时间点的 DNA 杂交和实时 PCR 结果。两种方法对检测副溶血性弧菌和创伤弧菌的符合率分别为 901 管的 857（95%）和 903 管的 882（98%）。总体而言，实时 PCR 和 DNA 菌落杂交的符合率为 96%。实时 PCR 获得的结果与 DNA 菌落杂交的结果相当，但用于检测经加工的牡蛎中的弧菌时，分析时间显著缩短。
