title: Development of a real-time PCR melt curve assay for simultaneous detection of virulent and antibiotic resistant Salmonella.
abstract: Multiple drug resistance in Salmonella is an emerging problem in the area of food safety. Depending on the virulence and antibiotic resistance characteristics of the Salmonella strain, infections of varying severity could result. In this study, a multiplex melt curve real-time PCR assay for the detection of virulent and antibiotic resistance strains of Salmonella was developed with two primer sets. The first set targets the virulence gene, invasin (invA), and tetracycline (tetG), streptomycin (aadA2) and sulphonamide (sulI) antibiotic resistance genes, and the second set amplifies ampicillin (blaPSE,blaTEM) and chloramphenicol (floR) resistance genes. The multiplex assay was evaluated using 41 Salmonella strains and was further tested on eight different artificially inoculated food samples. The fluorescent DNA intercalating dye, SYTO9, generated high resolution melt curve peaks and, hence, was used for the development of the assay. This multiplex assay worked efficiently over a DNA concentration range of 20 ng-200 fg and showed a sensitivity of 290 CFU/mL with serially diluted broth cultures. The detection limit for un-enriched artificially inoculated food samples was 10(4) CFU/g, but an enrichment period of 6 h allowed for detection of 10 CFU/g of cells in the samples.
authors.affiliation: Food Science Program, 256 WCS Wing, Eckles Hall, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Prashant
authors.initials: P
authors.lastname: Singh
authors.affiliation: Food Science Program, 256 WCS Wing, Eckles Hall, University of Missouri, Columbia, MO 65211, USA. Electronic address: MustaphaA@missouri.edu.
authors.forename: Azlin
authors.initials: A
authors.lastname: Mustapha
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D012480:Salmonella Infections / Q000382:microbiology*; D037521:Virulence Factors / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D037521:Virulence Factors
keywords: Melt curve analysis; Multiple drug resistance; Real-time PCR; Salmonella