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.
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.
Institute of Biochemistry and Biophysics, P.O. Box 13145-1384, University of Tehran, Tehran, Iran.
Hadi
H
Ravan
Razieh
R
Yazdanparast
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005798:Genes, Bacterial; D057230:Limit of Detection; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011200:Poultry / Q000382:microbiology*; D012480:Salmonella Infections / Q000175:diagnosis; D019779:Salmonella enterica / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012703:Serotyping / Q000379:methods
D004269:DNA, Bacterial