Background The Clear Safety Listeria method utilizes polymerase chain reaction (PCR) amplification and targeted next-generation sequencing technology to detect Listeria species (L. monocytogenes, L. innocua, L. ivanovii, L. marthii, L. grayi, L. welshimeri, and L. seeligeri) in hot dogs and on selected environmental surfaces. Objective The aim was to validate the candidate method according to current AOAC guidelines. Method The candidate method was compared to the reference method for hot dogs and the environmental surfaces. The method was also evaluated for inclusivity and exclusivity using 50 inclusivity strains and 30 exclusivity strains for each reported target. Product consistency and stability was tested and robustness was evaluated with changes in enrichment temperature, volume of sample treatment, and aliquot volume for PCR. Results The candidate method demonstrated no statistically significant differences using the probability of detection model between candidate and reference methods or between presumptive and confirmed results for all environmental surfaces and hot dogs. Additionally, the candidate method detected all inclusivity organisms and excluded all exclusivity organisms for each reported target. Product lots were shown to be consistent and data supported the kit’s shelf life. Finally, the robustness study demonstrated no statistical differences when the volume of sample or the aliquot volume for PCR was altered. Increasing the incubation temperature to 37 ± 1 °C resulted in greater recovery of L. monocytogenes as compared to 35 ± 1 °C and 30 ± 1 °C. Conclusions The Clear Safety Listeria method is statistically equivalent to the reference methods for the detection of L. monocytogenes and Listeria spp. in hot dogs and on selected environmental surfaces. Highlights The Clear Safety Listeria method is an automated, highthroughput NGS-based method capable of detecting Listeria species in the hot dog and environmental samples within 28h.

中文翻译：

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验证用于检测热狗和环境表面基质中李斯特菌的明确安全李斯特菌方法：AOAC 性能测试方法SM 091901

背景 Clear Safety 李斯特菌方法利用聚合酶链式反应 (PCR) 扩增和靶向二代测序技术来检测李斯特菌种类（单核细胞增生李斯特菌、无害李斯特菌、伊万诺氏菌、马氏李斯特菌、灰色李斯特菌、威尔士李斯特菌、和 L. seeligeri）在热狗和选定的环境表面上。目的 目的是根据当前的 AOAC 指南验证候选方法。方法 将候选方法与热狗和环境表面的参考方法进行比较。对于每个报告的目标，还使用 ​​50 个包容性菌株和 30 个排他性菌株评估了该方法的包容性和排他性。测试了产品的一致性和稳定性，并通过富集温度、样品处理体积和 PCR 等分体积的变化来评估稳定性。结果候选方法使用检测模型的概率在候选方法和参考方法之间或在所有环境表面和热狗的推定结果和确认结果之间没有显示出统计学上的显着差异。此外，候选方法检测到所有包容性生物并排除每个报告目标的所有排他性生物。产品批次显示一致，数据支持试剂盒的保质期。最后，稳健性研究表明，当样品体积或 PCR 的等分体积发生变化时，没有统计学差异。与 35 ± 1 °C 和 30 ± 1 °C 相比，将孵育温度提高到 37 ± 1 °C 导致单核细胞增生李斯特菌的回收率更高。结论 Clear Safety 李斯特菌方法在统计学上等同于检测单核细胞增生李斯特菌和李斯特菌属的参考方法。在热狗和选定的环境表面上。亮点 Clear Safety Listeria 方法是一种基于 NGS 的自动化、高通量方法，能够在 28 小时内检测出热狗和环境样品中的李斯特菌种类。