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.

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.

QCM-based rapid detection of PCR amplification products of Ehrlichia canis.

Ehrlichia canis is an intracellular parasitic bacterium and arthropod-borne pathogen that receives growing attention, because it leads to increasing morbidity and mortality in animals. It does so by causing canine monocytotropic ehrlichiosis (CME). Infected canines may lack obvious clinical signs and stay in chronic stage. Herein we report a rapid screening method based on PCR assay combined with quartz crystal microbalance (QCM) to design a DNA sensor for detecting E. canis in early stages of infection. The test relies on DNA amplification of target nucleotide sequences via PCR followed by detecting DNA-DNA hybridization using QCM. The approach did not result in any cross-hybridization toward other blood bacteria or parasites in dogs, such as Anaplasma platys, Babesia canis and Trypanosoma spp, but turned out selective for the target species. The limit of detection of QCM was as low as 4.1 × 109 molecules/µl of 289 bp E. canis PCR products corresponding to 22 copy numbers/µl of E. canis. Furthermore, the technique is also simple, does not require complicated equipment and can in principle be reused.