Division of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD 20708, USA; Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA.
Division of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD 20708, USA; Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA.
Food Microbiol. 2015 Apr;46:485-493. doi: 10.1016/j.fm.2014.09.011. Epub 2014 Sep 28.
Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study.
快速、可靠、稳健地检测农产品中的沙门氏菌仍然是一个挑战。在本研究中，环介导等温扩增（LAMP）技术与实时定量 PCR（qPCR）技术进行了全面比较，用于检测不同农产品（哈密瓜、胡椒和几种生菜、芽菜和番茄）中的多种沙门氏菌血清型。为了模拟实际污染事件，将低浓度（1.1-2.9 CFU/25 g）的代表十种血清型的单一沙门氏菌菌株接种到农产品表面，然后在 4°C 下老化 48 小时后进行测试。还比较了四种使用农产品增菌肉汤的 DNA 提取方法。在用于评估检测特异性的 178 株菌（151 株沙门氏菌和 27 株非沙门氏菌）中，没有观察到假阳性或假阴性结果。LAMP 在纯培养物中的检测限为 1.8-4 CFU/反应，在未增菌的农产品中为 10(4)-10(6) CFU/25 g（即 10(2)-10(4) CFU/g），与 qPCR 获得的结果相当。经过 6-8 小时的增菌，LAMP 和 qPCR 都能在所有农产品中检测到除芽菜外的低浓度、不同血清型的沙门氏菌。在四种 DNA 提取方法中，PrepMan Ultra 样品制备试剂的效果最好。进一步验证后，LAMP 可能成为农产品中常规沙门氏菌检测的一种有价值的工具。无论是使用 LAMP 还是 qPCR，在芽菜中检测沙门氏菌的困难都需要进一步研究。
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