Purpose: This study evaluated inclusivity/exclusivity, LDi, and performance of a novel qPCR-based method for detecting this large family of bacteria in different infant formula and associated ingredients.
Methods: The test includes five steps: enrichment, Free DNA Removal Solution (FDRS), DNA extraction, real-time PCR, and data interpretation. For inclusivity/exclusivity testing, 282 certified strains at ~105 CFU/ml or DNA were tested (including 248 belonging to the EB family). The LDi was determined by using a 10-fold serial dilution of a Salmonella spp. strain from equivalent 7.4 x 106 to 0.74 CFU/ml. To evaluate the method sensitivity, 17 different matrices (12 milk powders, 3 with probiotics, and 5 ingredients) were inoculated at 5 CFU/ml (or not). All samples were culture confirmed following the ISO 21528-1:2004 method. Cq values for both EB and internal control targets were analyzed.
Results: The assay demonstrated 100% inclusivity for EB and 100% exclusivity for non-EB with the collection of strains evaluated. LDi results were confirmed to 68 CFU/ml. For effectiveness testing, concordance to culture confirmation was 100% for all PCR assays, even on probiotic formula enriched with vancomycin.
Significance: This study indicates that the iQ-Check® Enterobacteriaceae in combination with the FDRS is a rapid and sensitive method for detecting EB in dairy infant formula and ingredients prone to the presence of dead cells. Test kit results demonstrated no significant difference when compared with the reference culture method.