USE OF GRAM‐NEGATIVE MEDIUM AND IMMUNOMAGNETIC SEPARATIVE METHOD FOLLOWED BY MULTIPLEX POLYMERASE CHAIN REACTION FOR THE DETECTION OF ENTEROHEMORRHAGIC ESCHERICHIA COLI AND SALMONELLA SPP. WITH GREAT CELL COUNT DIFFERENCE IN FOOD SAMPLES

Abstract

ABSTRACTContamination of different bacterial species with great cell count difference in food samples may affect the efficacy of multiplex polymerase chain reaction (mPCR) detection of these organisms. The purpose of this study was to improve the efficiency of mPCR detection of two pathogenic bacterial species, i.e., Salmonella and Shiga‐like toxin I Escherichia coli (SLTI EC), with great cell count difference in food samples. Without the enrichment step, when the cell count ratio of these two species was higher than 103, the bacteria presenting the lower number might go undetectable. For beef and milk samples with great ratios of SLTI EC/Salmonella, such as 103–105/100–101, if gram‐negative (GN) broth was used for the pre‐enrichment step, both organisms were detectable by mPCR. However, for samples with great ratios of Salmonella/SLTI EC, after enrichment with GN broth, an immunomagnetic separation (IMS) step using equimolar mixture of anti‐Salmonella and anti‐enterohemorrhagic Escherichia coli immunobeads had to be performed prior to mPCR to allow obvious results for both organisms. Therefore, although GN is a selective medium for both Salmonella and E. coli, depending on the ratios of the two target organisms in food samples, an IMS step may be required to allow the simultaneous PCR detection of both organisms.PRACTICAL APPLICATIONSAlthough multiplex polymerase chain reaction (mPCR) method allows the simultaneous detection of different target organisms, if the ratios of the cell counts for these target organisms were great, the PCR results for target organisms with lower counts will be obscured. Using gram‐negative broth for pre‐enrichment followed by mPCR or pre‐enrichment and immunomagnetic separation (IMS) followed by mPCR, we were able to simultaneously detect enterohemorrhagic Escherichia coli and Salmonella in food samples even the cell count ratios of these two organisms were great. Similar approach could be used for the simultaneous detection of two or more bacterial species in different samples.