Validation Using Diverse, Difficult-to-Detect Salmonella Strains and a Dark Chocolate Matrix Highlights the Critical Role of Strain Selection for Evaluation of Simplified, Rapid PCR-Based Methods Offering Next-Day Time to Results

Abstract

Abstract: 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.

Keywords: Salmonella; AOAC; Chocolate; PCR; Rapid method; Validation.