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.

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.

Validation of the Modified Clear Safety Salmonella for Detection of Salmonella enterica in Selected Poultry and Pet Food Matrixes and on Stainless Steel: AOAC Performance Tested MethodSM 111802.

BACKGROUND: The Clear Safety Salmonella method was modified to improve sample preparation, PCR reagents, library preparation, flow cell quality control, library loading mix, priming mix, and sequencing kit reagents and steps. OBJECTIVE: To evaluate the modified Clear Safety Salmonella method (manual and automated) via independent and method developer validation studies according to current AOAC INTERNATIONAL Validation Guidelines. METHOD: Performance of the modified Clear Safety Salmonella method (manual and automated) was assessed for selectivity (using 105 inclusive and 30 exclusive strains), probability of detection in matrixes, product consistency, stability, and robustness. The modified Clear Safety Salmonella method was compared with the appropriate reference method for Salmonella detection on 4 inch × 4 inch stainless steel environmental surfaces, and in chicken carcass rinse (30 mL), raw ground chicken (375 g), dry pet food (375 g), and ready-to-eat deli turkey breast (375 g). RESULTS: The modified Clear Safety Salmonella method (manual and automated) demonstrated no statistically significant differences between the candidate and reference method probability of detection or between the presumptive and confirmed results for all target food matrixes and the stainless steel surface. Additionally, the modified method (manual and automated) detected all 105 inclusivity organisms and excluded all 30 exclusivity organisms. The product consistency and kit stability studies showed no statistical differences between lots or over the term of the kit's shelf life. In robustness studies, changes in enrichment time, diluted sample volume, and sample volume for PCR did not show any statistical difference in terms of assay performance. CONCLUSIONS: The modified Clear Safety Salmonella method (both manual and automated) is statistically equivalent to or better than the reference methods. HIGHLIGHTS: The Clear Safety Salmonella method utilizes PCR amplification and targeted next-generation sequencing technology to selectively detect Salmonella enterica.