P2-170 Evaluation of a Novel Method for Detection of Enterobacteriaceae in Dairy Infant Formula Using Real-time PCR

Tuesday, July 11, 2017
Exhibit Hall (Tampa Convention Center)
Laurent Jain , Bio-Rad Laboratories , Marnes-la-Coquette , France
André Quintanar , Bio-Rad , Hercules , CA
Jean-Philippe Tourniaire , Bio-Rad Laboratories , Hercules , CA
Sophie Pierre , Bio-Rad Laboratories , Hercules , CA
Jean-François Mouscadet , Bio-Rad , Marnes-la-Coquette , France
Introduction: Enterobacteriaceae (EB) are found in many food matrices; among them dairy infant formula and related environmental sources. Their absence is required since they can cause serious illness in babies. While isolation by culture can be a labor intensive process, a real-time PCR method could allow rapid qualitative detection, especially in the presence of competing free DNA and flora.

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.