Development and evaluation of a loop-mediated isothermal amplification method in conjunction with an enzyme-linked immunosorbent assay for specific detection of Salmonella serogroup D

Abstract

Loop-mediated isothermal amplification in conjunction with enzyme-linked immunosorbent assay (LAMP-ELISA) provides a sensitive, specific and cost-effective method for detection of etiological causes of infections. The present study developed a reliable LAMP-ELISA diagnostic kit for identification of Salmonella serogroup D strains and evaluated its potential use in the detection of Salmonella serovars Enteritidis and Typhi. The LAMP-ELISA assay used a serogroup D/A-specific primer set to amplify a region of the prt gene, followed by hybridization of the digoxigenin-labeled products to a highly specific oligonucleotide probe for exact identification of serogroup D serovars. Among the bacteria tested, a positive reaction was only observed for strains belong to Salmonella serogroup D. The detection limit of the LAMP-ELISA assay was 4 CFU per tube, which was lower than PCR-ELISA assay with the same target gene (50 CFU per tube). Finally, the technique was successfully applied to an artificially contaminated meat sample with a detection limit 10(3) CFU mL(-1), which was 10 times more sensitive than PCR-ELISA. Overall, the LAMP-ELISA assay could be used as a sensitive alternative method to PCR-ELISA for the specific detection of Salmonella serogroup D serovars in routine food microbiology or clinical laboratories worldwide.