Russo Pasquale, Botticella Giuseppe, Capozzi Vittorio, Massa Salvatore, Spano Giuseppe, Beneduce Luciano
Department of Agriculture, Food and Environmental Sciences, University of Foggia, Via Napoli 25, 71122 Foggia, Italy.
Biomed Res Int. 2014;2014:608296. doi: 10.1155/2014/608296. Epub 2014 May 15.
In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g(-1) could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 10(1) CFU g(-1) after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype.
在本研究中，我们开发了一种用于快速、可靠地检测和定量加工最少的蔬菜中单核细胞增生李斯特菌和大肠杆菌O157:H7的MPN定量实时PCR（MPN-qPCR）方法。为了验证所提出的技术，将结果与传统的MPN法以及随后的表型和生化检测方法进行了比较。当单核细胞增生李斯特菌和大肠杆菌O157:H7人工接种到鲜切蔬菜中时，两种病原体在48小时内都能检测到低至1 CFU g(-1)的浓度。对于单核细胞增生李斯特菌和大肠杆菌O157:H7，仅qPCR在富集2小时后检测限为10(1) CFU g(-1)。由于加工最少的即食蔬菜保质期非常短，我们的方法有可能解决微生物分析时间持续减少的问题，从而更好地进行质量控制管理。此外，在两个生产工厂中，从原材料接收到保质期早期阶段，对混合沙拉样品和鲜切瓜中这两种病原菌的出现情况进行了监测。未发现有样品被单核细胞增生李斯特菌污染。一份生混合沙拉样品被检测出H7肠出血性血清型呈阳性。
