Development of a multiplexed PCR assay combined with propidium monoazide treatment for rapid and accurate detection and identification of three viable Salmonella enterica serovars

Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes (Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella. Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 x 10(6) CFU/ml in pure culture or 1 x 10(6) CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 10(2) CFU/ml (4.3 x 10(2) CFU/ml for S. Typhimurium, 3.7 x 10(2) CFU/ml for S. Paratyphi B. 7.2 x 10(2) CFU/ml for S. Typhi) in pure culture and 10(3) CFU/g (4.3 x 10(3) CFU/g for S. Typhimurium, 3.7 x 10(3) CFU/g for S. Paratyphi B, 7.2 x 10(3) CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S. Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce. (C) 2012 Elsevier Ltd. All rights reserved.