Designing species-specific primers for various bacteria found in foods

This study aimed to design species-specific primers for the rapid, specific, and sensitive detection of pathogenic bacteria that are critically important for food safety, and to evaluate their effectiveness under laboratory conditions using the Polymerase Chain Reaction (PCR) technique. The development of such molecular diagnostic methods is of great importance for preventing foodborne illnesses, protecting public health, and ensuring the sustainability of food industry reliability. The study focused on designing primers for significant bacterial species, particularly Salmonella spp., Escherichia coli, Listeria spp., and Bacillus spp., and experimentally evaluated their specificity, sensitivity, and amplification efficiency. In primer design, conserved regions such as the 16S rRNA gene were targeted alongside species-specific genes (e.g., bceT, cyt/hk, prs, iap, iroN, and uspA). Optimization was performed by testing different annealing temperatures and PCR conditions to identify primers yielding successful results. In this study, the bceT-specific primers designed for Bacillus spp. successfully amplified the target at an annealing temperature of 51°C. However, the cytK and tuf gene primers for the same bacteria failed to produce the expected results, indicating no amplification of the target DNA. For Salmonella Typhimurium, varying results were obtained for different genes. While the iroN gene amplified effectively at 54°C, the bigA gene performed optimally at 57°C and 59°C. The iacP gene exhibited an interesting outcome which indicated that standard PCR failed to yield results, but successful amplification was achieved using gradient PCR. For Listeria monocytogenes, the iap and prs gene primers did not produce amplification under any tested experimental conditions. This suggests the need for primer sequence reoptimization or indicates potential genetic polymorphisms in the target regions. In contrast, the uspA gene primers for Escherichia coli performed successfully following the protocol specified in the literature, confirming this gene's reliability as a species-specific molecular marker. The findings demonstrate that while some of the designed primer sets were effective in identifying foodborne pathogens, others require reoptimization of physicochemical parameters.