Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 x 10³ or 5.13 x 10⁴ CFU/g for VBNC E. coli O157:H7 and 1.05 x 10⁴ or 1.05 x 10⁵ CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.

IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

大肠杆菌O157：H7和肠沙门氏菌是与新鲜农产品相关的食源性暴发的主要原因。两种物种都可以进入“可行但不可培养”（VBNC）状态，从而无法使用常规的基于培养的方法或分子方法进行检测。在这项研究中，我们评估了单叠氮化丙啶定量PCR（PMA-qPCR）分析方法和结合PMA和环介导的等温扩增（LAMP）的新颖方法，用于检测和定量新鲜VBNC大肠杆菌O157：H7和肠炎沙门氏菌。生产。PMA-LAMP测定靶向的性能WZY的基因的大肠杆菌O157：H7和AGFA的基因沙门氏菌并比较了纯培养物和加标番茄，生菜和菠菜中PMA-qPCR测定的性能。在特异性测试中未观察到交叉反应。在纯培养物中，代表PMA-LAMP的检出限（LOD）的值对于大肠杆菌O157：H7为9.0 CFU /反应，对于肠炎链球菌为4.6 CFU /反应，分别为5.13 x 10 ³或5.13 x 10 ⁴ CFU /克为VBNC大肠杆菌O157：H7和1.05 X 10 ⁴或1.05 X 10 ⁵ CFU / g的为VBNC沙门氏菌在新鲜农产品中，其结果可与通过PMA-qPCR获得的结果相媲美。标准曲线显示相关系数在0.925至0.996之间，表明PMA-LAMP具有很好的定量能力，可以确定VBNC状态下两种细菌的​​种群。完成PMA-LAMP分析所需的时间非常可观（30分钟对1小时），并且灵敏度和定量能力可与PMA-qPCR分析相媲美。PMA-LAMP是一种快速，灵敏且稳定的方法，用于检测和定量新鲜农产品中的VBNC大肠杆菌O157：H7和小肠链球菌。

重要信息VBNC致病细菌不会在常规微生物培养基上繁殖，因此对食品工业构成潜在风险，因此可以逃避常规平板检测中的检测。这两种大肠杆菌O157：H7和沙门氏菌已报道在一系列的环境胁迫条件进入VBNC状态和有利条件下复苏，并且是人类感染的潜在原因。在这项研究中开发的PMA-LAMP方法提供了一种快速，灵敏和特定的方法来测定VBNC大肠杆菌O157：H7和肠炎链球菌的水平在这种状态下，可以潜在地降低食用与被肠道病原体污染的新鲜农产品有关的风险。PMA-LAMP可进一步应用于田间研究，以增进我们对新鲜农产品收获前和收获后VBNC病原体命运的了解。