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    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. The aim was to validate the candidate method according to current AOAC guidelines. 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. 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. 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. 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. © AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email:


    Stephanie Pollard, Atul K Singh, Andrew Lin, James Maloney, Anay Campos, Ramin Khaksar, Benjamin Bastin, Wesley Thompson, M Joseph Benzinger, James Agin. Validation of the Clear Safety Listeria Method for Detection of Listeria Species in Hot Dogs and on Environmental Surface Matrixes: AOAC Performance Tested MethodSM 091901. Journal of AOAC International. 2022 Feb 04;105(1):211-229

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    PMID: 34181026

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