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 Validation Guidelines. METHODS 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 to the appropriate reference method for Salmonella detection on a 4" × 4" 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 (dPODC) or between the presumptive and confirmed results (dPODCP) 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.