Purpose: Development and validation have been performed for this workflow, which is the first of its kind in the market, and allows food safety professionals to utilize this pooling approach prior to detecting Listeria by Real-time PCR.
Methods: The core of this isolation technology is the automated purification of all Listeria species of significance by antibody-conjugated magnetic beads specific for Listeria. The captured bead-bound bacteria are then lysed, and the supernatant is added to an AOAC- and AFNOR-validated lyophilized MicroSEQ® Listeria spp. (or Listeria monocytogenes) Real-time PCR assay. By combining the specificity of antibody-based capture and the sensitivity of Real-time PCR, we endeavored to reliably detect 1 CFU in 25-g food samples, as well as in swab- and sponge-based environmental samples.
Results: A wide variety of over 30 different sample types (n > 300 total samples) were tested during internal validation. This workflow correctly identified 96% of all samples against the Reference Method by Real-time PCR, and 98% by selective agar plating.
Significance: The ability to pool individual samples, in addition to the ease of use of this workflow, enables the processing of hundreds of samples per hour at a fraction of the cost of platforms that do not accommodate a pooled sample format, thus creating an economic benefit to food producers by providing a workflow that is able to rapidly and inexpensively screen for rare contamination events in both the environment and food products. This work demonstrates that one can use PCR technology to detect pathogens without the costs and labor associated with the traditional one-sample-per-assay relationship.