Mpl-Gene-Based Loop-Mediated Isothermal Amplification Assay for Specific and Rapid Detection of Listeria monocytogenes in Various Food Samples.

Abstract

Listeria monocytogenes represents a high risk in food and can trigger potentially fatal listeriosis. The objective of this study was to detect L. monocytogenes in food using the LAMP method in a fast, specific, sensitive manner and thus to preventively test food for the presence of the target species. The reaction was performed and established using the portable real-time fluorometer Genie® II (OptiGene Ltd., Horsham, United Kingdom). In this new assay, six LAMP primers targeted the mpl-gene sequence of L. monocytogenes. A total of 148 different isolates, including 105 L. monocytogenes and 43 non-L. monocytogenes strains, were tested. Analytical sensitivity was determined based on different DNA- and cell concentrations. The detection limit with a detection rate of 100% was 5 pg of DNA or 275 colony-forming units (CFU) per reaction. Artificially contaminated minced beef and grated mozzarella were also tested. The assay was 100% successful to detect an initial bacterial contamination of 0.4-4 CFU g-1 food after 24 h enrichment in half-Fraser broth. Finally, natively contaminated samples were tested in comparison to the microbiological reference method and real-time polymerase chain reaction. Native sample testing revealed 100% consistent findings between LAMP and the standard culture method after first enrichment for 24 h. In addition, a rapid colony-confirmation method was established that enabled reliable identification of L. monocytogenes isolates on different selective culture media using a simplified DNA extraction by boiling. This study showed that the developed assay was able to determine whether a food is safe with respect to the food-safety criteria of 100 CFU per gram, according to standards of the European Union, for L. monocytogenes and provided faster results than the cultural reference method.