Novel Immunoassay Pathogen Detection Method for Listeria spp. in Food and Environmental Samples

Introduction: Immunoassay-based pathogen detection methods are typically comprised of two selective enrichment steps prior to detection, resulting in a longer time to result than some alternative methods. We have developed a next generation Listeria spp. immunoassay, which provides negative or presumptive positive results within 26 to 29 hours.

Purpose: The aim of our study was to shorten the total method time of our existing Solus Listeria immunoassay by improving the enrichment and immunoassay performances.

Methods: The resulting Listeria immunoassay was performed following enrichment of a 25 g sample in 225 ml selective broth for 26 hours at 30°C. Analytical sensitivity was determined with enumerated heat-killed cultures at 5×10⁵, 1×10⁴, and 1×10⁶ CFU/ml. Inclusivity and exclusivity was tested with 10 Listeria spp. and 38 non-Listeria spp. Samples spiked at 1 to 10 CFU Listeria per 25 g. Detection of the target organism was tested within naturally contaminated matrices (ready-to-eat and ready-to-reheat, meat, dairy, vegetable and seafood products, ingredients, feed products and environmental samples).

Results: The analytical sensitivity of the assay was calculated to be approximately 10⁴ to 10⁵ CFU/ml. All food associated Listeria spp. were detected and a panel of 38 non-Listeria spp. gave negative results indicating satisfactory inclusivity and exclusivity. Three hundred and one naturally contaminated food samples were tested from the categories above. Sixty-four samples were found to contain Listeria by the EN ISO 11290 reference method and 62 of 64 samples were positive by the immunoassay and subsequent confirmation from the alternative enrichment protocol. No false positive results were observed.

Significance: The resulting immunoassay retains the testing efficiency benefits of the original assay, which, when used in conjunction with liquid handling automation, enables processing of large numbers of samples. Increased sensitivity enables a faster time to result, which is clearly beneficial for rapid turnaround of food and environmental test samples when a contamination source needs to be identified and eliminated.