A RAPID, SENSITIVE AND AUTOMATED-METHOD FOR DETECTION OF SALMONELLA SPECIES IN FOODS USING AG-9600 AMPLISENSOR ANALYZER

The AG-9600 AmpliSensor Analyzer is an automated fluorescence-based sy stem for detection of polymerase chain reaction (PCR) produces, The pr inciple of the AmpliSensor PCR assay involves amplification-mediated d isruption of a fluorogenic DNA signal duplex (AmpliSensor) that is hom ologous to a target sequence within a 284-bp amplified fragment of the Salmonella invA gene, Since the assay is homogenous, the data can be obtained by direct measurement of fluorescence of the amplification mi xture, The accumulation of the amplified product, reflected by the flu orescence index, is monitored cycle by cycle by the AG-9600 Analyzer. The detection limit of the assay was less than 2 colony-forming units (cfu) per PCR reaction using a pure culture of Salmonella typhimurium. In post-spiking experiments in which Salmonella was added to the over night pre-enriched samples (chicken carcass rinses, ground beef, groun d pork and raw milk), the detection limit of the assay was 2-6 cfu per PCR reaction. In pre-spiking experiments in which Salmonella was adde d to the samples prior to overnight pre-enrichment, the detection limi t was less than 3 cfu per 25 g or 25 ml of food, The assay was up to 2 orders of magnitude more sensitive than detection by ethidium bromide -stained agarose gel electrophoresis. To further evaluate assay perfor mance, 54 naturally contaminated chicken carcass rinses, 65 raw milk a nd six ground pork samples were tested in the study. Thirty-eight Salm onella-positive samples confirmed by the Modified Semi-solid Rappaport -Vassiliadis (MSRV) culture assay were found positive using the AmpliS ensor assay. Two chicken carcass rinses found positive using the assay were MSRV-negative. In addition, relative quantification using the Am pliSensor assay was linear up to 3 logs of initial target concentratio n in artificially contaminated food samples.