NOVEL MULTIPLEX PCR TO SPECIFICALLY DETECT BACTERIAL FOODBORNE PATHOGENS
Keywords:
Multiplex PCR, foodborne pathogens, optimization, target bacteriaAbstract
Bacterial foodborne pathogens prevalent in poultry, especially Escherichia coli, Salmonella spp., Shigella spp., and Listeria monocytogenes, have been reported in many countries including Thailand. Rapid methods for identification and detection of these dominant foodborne pathogens are still required. In our study, multiplex polymerase chain reaction (m-PCR) was developed for detecting multiple bacterial foodborne pathogens. Specific genes for the m-PCR primers were screened and selected. m-PCR targeting the uspA, fimY, ipaH, and prfA gene was used to detect E. coli, Salmonella spp., Shigella spp., and L. monocytogenes, respectively. The optimum conditions for the m-PCR reaction were found to be primer concentrations of 0.02 μM ipaH, 0.036 μM fimY, 0.06 μM uspA,0.12 μM prfA, and 0.4 μM 16S rRNA gene (used as internal control) for at least 10 ng of each bacterial total genomic DNA; and 52oC was the annealling temperature. The expected PCR products of 884, 489, 422, and 398 bp were obtained from specific amplification of E. coli, Salmonella spp., Shigella spp., and L. monocytogenes, respectively, of the reference strains and strains isolated from fresh chicken intestine. Cross amplification from non-target bacteria which have been frequently found in enrichment culture were not detected. These results indicated that the developed m-PCR could be used to detect multiple foodborne pathogens with no cross-reactivity with the non-target bacteria found in the enrichment culture. Alternatively, this method could also be used to identify the presumptive colonies of interest on selective agar with considerable timesaving when compared with the biochemical characterization of the conventional method.
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