THE EVALUATION OF A FLUOROGENIC POLYMERASE CHAIN-REACTION ASSAY FOR THE DETECTION OF SALMONELLA SPECIES IN FOOD COMMODITIES

The TaqMan(TM) LS-50B PCR Detection System facilitates the automated a nd direct detection of polymerase chain reaction (PCR) products. The s ystem employs the 5' nuclease activity of Taq DNA polymerase to hydrol yse a Salmonella specific internal fluorogenic probe for monitoring th e amplification of a 287-bp region of the Salmonella invA gene. Using the fluorogenic 5' nuclease assay, 164 Salmonella strains representing all the subspecies of Salmonella enterica were detected while over 50 non-Salmonella strains were not detected. The detection limit of the assay was two colony forming units (cfu) per PCR reaction when a pure culture of S. typhimurium was used. Six protocols for the isolation of PCR-amplifrable DNA were evaluated using chicken carcass rinses, grou nd beef, ground pork and raw milk contaminated with Salmonella. Of the six DNA isolation protocols, a modified sample preparation protocol u sing the EnviroAmp kit was chosen for subsequent studies because it wa s reliable, easy to use and efficient for the isolation of PCR-amplifi able DNA from foods. A detection limit of 3-7 cfu per PCR reaction was obtained using food samples that were pre-enriched overnight and then inoculated with Salmonella. The detection limit was below 3 cfu/25 g or 25 ml when foods inoculated with Salmonella were pre-enriched overn ight. Naturally contaminated foods (50 chicken carcass rinses and 60 r aw milk samples) were examined using both the fluorogenic 5' nuclease assay and a modified semi-solid rappaport vassiliadis (MSRV) culture m ethod. Thirty four of the 110 samples tested were Salmonella-positive and 74 were Salmonella-negative by both the 5' nuclease assay and the MSRV method. Two samples were Salmonella-positive by the 5' nuclease a ssay, but negative by the MSRV method. The correlation between the 5' nuclease assay and the MSRV method was over 98%. (C) 1997 Elsevier Sci ence B.V.