Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat

Enrichment cultivation in detection of food-borne Salmonella

“…Two reporter systems, the DNA binding dye technology (intercalating SYBR Ò Green assay) and the 5 0 nuclease assay (TaqMan Ò chemistry), have been particularly popular in the realtime PCR analysis of Salmonella (Levin, 2009;Postollec, Falentin, Pavan, Combrisson, & Sohier, 2011), but other detection systems such as Scorpion probes (Reynisson, Josefsen, Krause, & Hoorfar, 2006) and Molecular beacons (Chen, Martinez, & Mulchandani, 2000) have also been used for the purpose. Real-time PCR together with enrichment cultivation has been applied for the detection of Salmonella in various food matrices, such as eggs Malorny, Bunge, & Helmuth, 2007), tomatoes (Warren, Yuk, & Schneider, 2007), and different meats (McGuinness et al, 2009;O'Regan et al, 2008). Alternative amplification-based nucleic acid detection methods have also been developed for food-borne Salmonella, including isothermal nucleic acid sequencebased amplification (NASBA) (D'Souza & Jaykus, 2003), and loopmediated isothermal amplification (LAMP) (Ye et al, 2011).…”

Development and preliminary validation of a real-time RT-PCR based method targeting tmRNA for the rapid and specific detection of Salmonella

“…Following incubation, 100 μl of preenrichment culture was transferred to 10 ml RVS and incubated at 42°C for 6 h (McGuinness et al, 2009). For RNA isolations, 8 ml of Rappaport-Vassiliadis Soya peptone (RVS; Oxoid) culture was centrifuged (4200 ×g for 10 min) and the cell pellet washed in 8 ml phosphate buffered saline (PBS; Oxoid).…”

“…the real-time PCR reverse primer [FPR-5′-GAGTTGAACCCGCGTC-3′; (McGuinness et al, 2009)] as per manufacturers' instructions. The sample was either stored at −20°C or used immediately as template for real-time PCR.…”

“…Each of these methods comprise of a shortened culture enrichment followed by a real-time PCR detection assay. Additional real-time PCR based methods for the detection of Salmonella in food have been reported (Malorny, Bunge, & Helmuth, 2007;McGuinness et al, 2009;O'Regan et al, 2008) however a limited number of studies have been published which describe the detection of Salmonella by real-time reverse transcriptase PCR (real-time RT-PCR) (Miller, Draughon, & D'Souza, 2010;Techathuvanan, Draughon, & D'Souza, 2010).…”

“…In a previous study by McGuinness et al (2009), a real-time PCR based method for the specific detection of Salmonella on fresh meat was developed and validated following requirements as outlined in the ISO 16140 standard: "Microbiology of food and animal feeding stuffs -Protocol for the validation of alternative methods" (Anonymous, 2003). This method combined a modified culture enrichment step with real-time PCR.…”