Area of Molecular Biology and Biotechnology, ANFACO-CECOPESCA, Ctra Colegio Universitario No 16, Vigo 36310, Pontevedra, Spain.
Food Microbiol. 2010 Feb;27(1):122-31. doi: 10.1016/j.fm.2009.09.004. Epub 2009 Sep 15.
In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.
在这项工作中，设计并验证了一种用于检测鱼类和海鲜中最常见食源性病原体弧菌属物种的顺序多重 PCR 系统（霍乱弧菌、副溶血性弧菌、创伤弧菌、溶藻弧菌和拟态弧菌）。该方法采用分级方式运作，由终点多重 PCR 检测研究物种的 DNA 存在，然后进行多重 PCR 和片段分析，以评估检测菌株的活力。如果需要鉴定血清群、生物型和/或毒力因子水平，则可以应用该方法的最终多重 PCR 步骤。在方法验证阶段，使用了 40 个商业鱼类和海鲜产品样本。从西班牙各个河口地区采集了 63 个海洋生物样本，包括虾、蟹、双壳类软体动物和鱼类，以筛查弧菌属的存在情况，其中 2 个贻贝样本检测出副溶血性弧菌阳性。总的来说，该方法具有很强的稳健性，易于在常规分子诊断实验室中应用，可用于监测和同时检测海鲜样本中的所有这些细菌病原体，减少其他分析方法的费用和时间消耗。
