Development of a duplex lateral flow dipstick test for the detection and differentiation of Listeria spp. and Listeria monocytogenes in meat products based on loop-mediated isothermal amplification.

ABSTRACT

Listeria spp. are a group of gram-positive bacteria consisting of 20 species. Among them, Listeria monocytogenes is one of the major species that infects humans since it contaminates raw fruits, vegetables, and many others food products. The conventional methods for the detection of Listeria spp. and L. monocytogenes are time-consuming, taking 5-7 days. Herein, a duplex lateral flow dipstick (DLFD) test combined with loop-mediated isothermal amplification (LAMP) was developed for the identification of Listeria spp. and L. monocytogenes within approximately 45 min with the optimized LAMP reaction times at 63 °C. Under the optimized conditions, the method detection limits (MDL) with reference to genomic DNA and pure culture were 900 femtograms (fg) and 20 cfu/mL, respectively. The LAMP-DLFD showed no cross-reactivity with eighteen - other pathogenic bacteria such as Salmonella spp., Staphylococcus aureus, Escherichia coli, Campylobacter coli, C. jejuni, Enterococcus faecalis, Vibrio cholerae, V. parahaemolyticus, Pseudomonas aeruginosa, Shigella dysenteriae, S. flexneri, Bacillus cereus, Lactobacillus acidophilus, L. casei and Pediococcus pentosaceus. Among 100 samples of food products, LAMP-DLFD demonstrated 100% accuracy when compared to other standard detection methods, such as ISO11290-1, enzyme-linked fluorescent assay (ELFA) technology (VIDAS) and PCR. In conclusion, LAMP-DLFD proved to be highly specific and sensitive assays for screening detection of Listeria spp. and L. monocytogenes.