Purpose: This study aimed to develop a Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) method for the detection of L. monocytogenes in food matrices.
Methods: Available, online rRNA databases were analyzed for probe design. The selected probe was synthetized and tested using 67 representative strains, for determination of sensitivity and specificity. Detection in food matrices was optimized, testing rich and selective broths, individually and in combination. A validation assay was performed in ground beef, ground pork, milk, lettuce, and cooked shrimp using two ranges of contamination; low (0.2 - 2 CFU/25g or mL) and high level (2 - 10 CFU/25g or mL). Samples were analyzed by PNA-FISH and ISO 11290.
Results: The designed probe, when coupled with a blocker probe (1:2 ratio), was highly sensitive and specific; 100% (88.6 - 100, 95% CI) and 93.1% (75,8 - 98.8, 95% CI) respectively. None of the tested universal and selective broths were able to increase L. monocytogenes to detectable levels by PNA-FISH at 24h, with the exception of One Broth Listeria (OBL) and University of Vermont Medium (UVM); however, the fluorescence signal obtained for those enrichments was weak. For the double enrichment steps, the best outcome was reached using OBL for 24h followed by OBL for 18h, even at low inoculum levels. This combined step was able to successfully detect L. monocytogenes in a variety of food matrices, with a detection limit of 0.5 CFU/25g (0.2 - 0.8, 95% CI), which was in line with ISO 11290.
Significance: The procedure, here described, is specific, sensitive, and able to detect L. monocytogenes, similarly to ISO 11290, while reducing the time of analysis to two days.