Department of Food Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York 14853.
Mars, Inc., 6885 Elm Street, McLean, Virginia 22101, USA.
J Food Prot. 2020 Aug 1;83(8):1374-1386. doi: 10.4315/JFP-20-066.
Modifications to pathogen detection kits to accomplish simplified protocols with reduced time to results may impact method performance, particularly when combining shortened enrichment times and simplified enrichment procedures. We used Salmonella detection in dark chocolate as a model to test the impact of different enrichment times (minimum and maximum validated times) and procedures on detection of low levels of difficult-to-detect Salmonella strains, for three PCR kits that were AOAC International Performance Tested Method certified for detection of Salmonella spp. in dark chocolate. Initial inclusivity studies with pure cultures showed that all three kits detected 70 of 70 Salmonella spp. strains at 1 log above the theoretical limit of detection, with some strains yielding later cycle threshold values or having variable detection among technical replicates, indicating reduced assay performance for these strains. Based on these data, we selected a S. enterica subsp. enterica serovar Poona strain as well as three non-subsp. enterica strains to test the ability of the three kits to detect Salmonella in dark chocolate inoculated at low levels (0.06 to 1.18 most probable number per 25 g). With primary enrichment in skim milk at 35°C, detection frequency for all assays did not significantly differ from the reference method for both the minimum and maximum validated enrichment times. However, a pilot study that used primary enrichment in buffered peptone water at 42°C yielded significantly fewer positive samples (13 of 80) than were obtained with the U.S. Food and Drug Administration Bacteriological Analytical Manual method using enrichment in skim milk at 35°C (40 of 80 positive samples); strains representing subsp. houtenae and salamae were detected in significantly fewer chocolate samples than enrichment with skim milk. Our data indicate that continued efforts to simplify rapid pathogen detection kits may reduce kit performance in a way that can only be detected with stringent evaluation protocols that are designed to identify kit failure modes.
为了简化方案并缩短结果时间，对病原体检测试剂盒进行修改可能会影响方法性能，尤其是在结合缩短的富集时间和简化的富集程序时。我们使用黑巧克力中的沙门氏菌检测作为模型，测试了不同的富集时间（最短和最长验证时间）和程序对检测低水平难以检测的沙门氏菌菌株的影响，这是三种经过 AOAC 国际性能测试方法认证的 PCR 试剂盒，用于检测黑巧克力中的沙门氏菌属。最初用纯培养物进行的包容性研究表明，所有三种试剂盒均能检测到理论检测限以上 1 个对数的 70 株沙门氏菌属菌株，其中一些菌株的循环阈值值较晚或在技术重复检测中存在可变检测，表明这些菌株的检测性能降低。基于这些数据，我们选择了一株 S. enterica subsp. enterica 血清型波恩沙门氏菌菌株以及三种非 subsp. enterica 菌株，以测试三种试剂盒检测黑巧克力中低水平（每 25 克 0.06 至 1.18 个最可能数）接种的沙门氏菌的能力。在 35°C 下用脱脂乳进行初次富集时，所有检测方法的检测频率与最短和最长验证时间的参考方法均无显著差异。然而，一项使用 42°C 缓冲蛋白胨水进行初次富集的初步研究的结果表明，与使用 35°C 下脱脂乳进行的美国食品和药物管理局细菌分析手册方法相比，阳性样品（80 个阳性样品中的 40 个）显著减少；与脱脂乳相比，亚群 houtenae 和 salamae 的菌株在巧克力样品中的检出率明显降低。我们的数据表明，简化快速病原体检测试剂盒的持续努力可能会降低试剂盒的性能，而这种降低只能通过旨在识别试剂盒故障模式的严格评估方案才能检测到。
