title: Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica.
abstract: The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.
authors.affiliation: Food Safety Department, Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland. evonne.mccabe@teagasc.ie
authors.forename: Evonne M
authors.initials: EM
authors.lastname: McCabe
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Burgess
authors.forename: Edel
authors.initials: E
authors.lastname: O'Regan
authors.forename: Sheila
authors.initials: S
authors.lastname: McGuinness
authors.forename: Thomas
authors.initials: T
authors.lastname: Barry
authors.forename: Séamus
authors.initials: S
authors.lastname: Fanning
authors.forename: Geraldine
authors.initials: G
authors.lastname: Duffy
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis; D005504:Food Analysis / Q000295:instrumentation / Q000379:methods; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D008460:Meat / Q000382:microbiology*; D008461:Meat Products / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis; D012478:Salmonella Food Poisoning / Q000517:prevention & control; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification*; D015534:Trans-Activators / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C102236:HilA protein, Salmonella; D012329:RNA, Bacterial; D015534:Trans-Activators