Abstract Shiga toxin-producing Escherichia coli (STEC), Salmonella spp. and Listeria monocytogenes are continuously reported as causative agents of great concern regarding food safety and widespread contamination in many food varieties. Therefore, their simultaneous detection may be advantageous in terms of cost, time and labor savings and simplicity. This study developed a new, simple platform of multiplex real-time polymerase chain reaction (mRT-PCR) for specific, sensitive and rapid detection of STEC, Salmonella spp. and L. monocytogenes in food. The single-tube mRT-PCR format was developed by combining an 18 h enrichment step in simultaneous enrichment broth, boiling based on DNA extraction assay and a mRT-PCR detection system based on melting curve analysis using a fluorescent dye (EvaGreen) for detection of the presence or absence of the three target bacterial pathogens in food samples. Three specific peaks were clearly detected with average melting temperatures of 84.52 ± 0.90 °C, 87.51 ± 0.54 °C and 79.32 °C ± 0.48 °C for STEC, Salmonella spp. and L. monocytogenes, respectively. The sensitivity and specificity of these newly developed mRT-PCR platforms were further investigated using artificially and naturally contaminated food samples. The relative sensitivity, relative specificity and relative accuracy were all 100%, with a detection limit of 1 cfu for each target pathogen in 25 g of food sample. The developed platform of EvaGreen-based single-tube mRT-PCR for detection of the three target pathogenic bacteria in food samples provided results of absence or presence within 20 h. The newly developed mRT-PCR platform in this study offers a promising approach for simple, rapid, sensitive, specific and accurate detection of the three target bacterial pathogens in food.

中文翻译：

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基于多重实时 PCR 的熔解温度曲线分析新单管平台，使用 EvaGreen 同时筛选检测产志贺毒素大肠杆菌沙门氏菌。和食物中的单核细胞增生李斯特菌

摘要产志贺毒素大肠杆菌（STEC），沙门氏菌属。单核细胞增生李斯特菌和单核细胞增生李斯特菌被不断报道为引起食品安全和许多食品品种广泛污染的高度关注的病原体。因此，它们的同时检测在成本、时间和劳力节省以及简单性方面可能是有利的。本研究开发了一种新的、简单的多重实时聚合酶链反应 (mRT-PCR) 平台，用于特异性、灵敏和快速检测 STEC、沙门氏菌。和食物中的单核细胞增生李斯特菌。单管 mRT-PCR 格式是通过在同时富集肉汤中结合 18 小时富集步骤开发的，基于 DNA 提取分析的沸腾和基于熔解曲线分析的 mRT-PCR 检测系统，使用荧光染料 (EvaGreen) 检测食品样品中三种目标细菌病原体的存在与否。明确检测到三个特定峰，平均解链温度分别为 84.52 ± 0.90 °C、87.51 ± 0.54 °C 和 79.32 °C ± 0.48 °C，用于 STEC、沙门氏菌属。和单核细胞增生李斯特菌，分别。使用人工和自然污染的食物样品进一步研究了这些新开发的 mRT-PCR 平台的敏感性和特异性。相对灵敏度、相对特异性和相对准确度均为 100%，25 g 食品样品中每种目标病原体的检测限为 1 cfu。开发的基于 EvaGreen 的单管 mRT-PCR 平台用于检测食品样品中的三种目标致病菌，可在 20 小时内提供不存在或存在的结果。本研究中新开发的 mRT-PCR 平台为简单、快速、灵敏、特异和准确检测食品中的三种目标细菌病原体提供了一种有前途的方法。