Cristiano D, Peruzy M F, Aponte M, Mancusi A, Proroga Y T R, Capuano F, Murru N
Department of Food Microbiology, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2, 8055 Portici, NA, Italy.
Department of Veterinary Medicine and Animal Production, University of Naples "Federico II", Via Delpino 1, 80137 Napoli, Italy.
Int J Food Microbiol. 2021 Sep 16;354:109321. doi: 10.1016/j.ijfoodmicro.2021.109321. Epub 2021 Jun 29.
Yersiniosis - the 4th most commonly reported zoonosis in the European Union - is caused by the consumption of food contaminated with the bacterium Yersinia enterocolitica. The number of human cases and contaminated food samples is probably underestimated since conventional molecular methods currently proposed for Yersinia enterocolitica detection proved to have several limitations. Critical issues associated with the detection of Yersinia enterocolitica in meat and/or meat product has already been investigated, whereas data on the possible limits of the molecular methods for Yersinia enterocolitica detection in vegetables are still lacking. According to ISO method (ISO 18867:2015), real-time polymerase chain reaction (rtPCR) should be adopted for Yersinia enterocolitica detection, even if it proved to be affected by some biases. Recently, Droplet Digital PCR (ddPCR) has been introduced as a useful tool to detect and quantify different pathogenic bacteria in complex food matrices. However, its potential application for Yersinia enterocolitica detection in vegetables has never been investigated before. In the present study two molecular platforms (rtPCR and ddPCR) were used to evaluate the pathogen's behaviour in experimentally contaminated leafy greens (Lactuca sativa L.) and to assess the rate of detection achievable after the incubation for eleven days at different temperatures. By comparing, noticeable differences emerged between the two technical approaches: only ddPCR allowed the detection of the pathogen in leafy greens when contaminated at low levels. Moreover, results of the present work highlighted the importance of length and temperature of incubation on the survival and/or the growth of Yersinia enterocolitica in vegetables: at 18 and 25 °C the concentration of the pathogen considerably decreases along incubation. Based on data, the use of rtPCR leads to an underestimation of the true prevalence of pathogenic Y. enterocolitica in vegetables, while temperature and time currently proposed for Y. enterocolitica (25 °C for 24 h), allow optimizing detection. To conclude, ddPCR may be undoubtedly proposed as a reliable alternative strategy for the quick detection of the pathogen in food samples.
耶尔森氏菌病是欧盟第四大最常报告的人畜共患病，它是由食用被小肠结肠炎耶尔森氏菌污染的食物引起的。由于目前提出的用于检测小肠结肠炎耶尔森氏菌的传统分子方法存在若干局限性，人类病例数和受污染食品样本数可能被低估了。与在肉类和/或肉类产品中检测小肠结肠炎耶尔森氏菌相关的关键问题已经得到研究，而关于分子方法检测蔬菜中小肠结肠炎耶尔森氏菌的可能限度的数据仍然缺乏。根据ISO方法（ISO 18867:2015），即使实时聚合酶链反应（rtPCR）被证明存在一些偏差，也应采用该方法检测小肠结肠炎耶尔森氏菌。最近，液滴数字PCR（ddPCR）已被引入作为检测和定量复杂食品基质中不同病原菌的有用工具。然而，其在蔬菜中小肠结肠炎耶尔森氏菌检测方面的潜在应用此前从未被研究过。在本研究中，使用了两种分子平台（rtPCR和ddPCR）来评估病原菌在实验污染的绿叶蔬菜（生菜）中的行为，并评估在不同温度下孵育11天后可实现的检测率。通过比较，两种技术方法之间出现了显著差异：只有ddPCR能够在低水平污染时检测出绿叶蔬菜中的病原菌。此外，本研究结果突出了孵育时间和温度对小肠结肠炎耶尔森氏菌在蔬菜中存活和/或生长的重要性：在18℃和25℃下，病原菌浓度会随着孵育时间大幅下降。基于这些数据，使用rtPCR会导致低估蔬菜中致病性小肠结肠炎耶尔森氏菌的真实流行率，而目前建议的小肠结肠炎耶尔森氏菌培养温度和时间（25℃培养24小时）有助于优化检测。总而言之，ddPCR无疑可被提议作为快速检测食品样本中病原菌的可靠替代策略。
