Direct triplex loop-mediated isothermal amplification assay for the point-of-care molecular detection of Salmonella genus, subspecies I, and serovar Typhimurium

Abstract

In this study, a novel triplex loop-mediated isothermal amplification (LAMP) assay was developed and applied to a model of representative foodborne pathogen, Salmonella, to investigate the performance of the assay with high rapidity, specificity and sensitivity, and its ability to detect a pathogen in contaminated food. Three sets of LAMP primers were designed for target genes in Salmonella genus, subspecies I, and S. Typhimurium. Multiple targets were differentiated by the simultaneous analysis of annealing curves in a single LAMP reaction. In a triplex LAMP assay, each primer amplifies the target genes only, without any cross-reactivity with non-target genes. The limit of detection of this assay was 2.5 pg of S. Typhimurium DNA. The assay was applied to chicken meat artificially inoculated with S. Typhimurium. To decrease the inhibitory effects of the food matrix on the LAMP reaction, proteinase K was used in sample preparation, and the sample was heated and used directly as a DNA template. A combination of the direct extraction method optimized for food with triplex LAMP was able to detect low levels of S. Typhimurium (6.4 × 101 CFU/g) without a pre-enrichment step. This novel triplex multiplex LAMP assay successfully identified Salmonella genus, subspecies I, and S. Typhimurium within 60 min from sampling to results. This is therefore a rapid, reliable, sensitive, specific, and easy molecular diagnostic tool for on-site pathogen detection and food safety testing.