A two step multiplex-semi nested Polymerase Chain Reaction assay (m-sn PCR) for the simultaneous identification of four major foodborne pathogens

We describe the development of a multiplex-seminested Polymerase Chain Reac tion assay (m-sn PCR) which enables the simultaneous identification of 4 ma jor foodborne pathogens Listeria monocytogenes, Salmonella spp., Campylobac ter jejuni/coli and enterohemorrhagic (eae gene positive) Escherichia coli The first step multiplex PCR alone is capable of detecting 10(3) to 10(4)cf u/ml (colony forming units) for each species. Using the second step semines ted PCR signal amplification we could lower the species-specific detection limits to 1-10 cfu/ml for E. coli, 1 cfu/ml for L. monocytogenes, 1-10 cfu/ ml for C. jejuni/coli and 1 cfu/ml for Salmonella spp. To test its applicability, the assay was applied to ovine raw milk samples. Out of 47 samples, 16 were tested positive for L. monocytogenes by both th e m-sn PCR and the Vidas(R) assay. Conventional microbiology confirmed pres ence of L. monocytogenes in 64% of the samples suspected. Additional pathog ens included in the assay were not identifyable in the raw milk samples.