Purpose: Multiplex PCR could be a reasonable solution to detect microbial contamination in food samples. However, multiplex PCR assays have several disadvantages, such as increased complexity from multiple primer sets, low amplification yield, variable efficiency on different templates and poor universal applicability caused by different annealing temperature requirements of multiple primer sets.
Methods: A universal multiplex PCR (UM-PCR) could overcome these kinds of problems and increase the specificity and sensitivity of a detection method. A UM-PCR was developed and used to simultaneously detect E. coli O157:H7 and Salmonella spp. in food samples.
Results: This UM-PCR method detected the presence of E. coli O157:H7 and non-O157 Shiga toxin-producing E. coli by targeting eae, stx1 and stx2. At the same time, Salmonella spp. could be detected by targeting invA and sgx in the same assay with a sensitivity of 104 CFU/ml. We could detect 7 major enterohemorrhagic strains including E. coli O157:H7 and 34 serotypes of Salmonella in an enriched sample of ground beef and of leafy green vegetables using a 1:1 and 1:3 inoculation, respectively.
Significance: UM-PCR greatly improves the range of applicability of a multiplex PCR assay. In addition, this method could be applied in other areas, such as the analysis of polymorphisms, quantitative assays for pathogens and the identification of bacterial species.