Harnessing bioinformatics to identify unique gene sequences tailored for the specific detection of Staphylococcus aureus by real-time PCR

Staphylococcus aureus is a major pathogen responsible for a wide range of foodborne illnesses. When the bacterial density of S. aureus reaches 105 CFU/mL, the pathogen begins producing enterotoxin - the main agent responsible for illness. Previous studies have predominantly focused on detecting enterotoxin or anti-biotic resistance strains. In recent years, real-time PCR (qPCR) targeting a specific gene has proven to be an efficient and precise tool for the early detection of this pathogen, even at very low bacterial concentrations, before toxin production begins. This study aimed to identify the most specific and suitable genetic marker for qPCR-based detection of S. aureus, harnessing bioinformatics tools to evaluate six candidate genes – sa442, nuc, femA, mecA,mecC, spA and coa. Conserved genes and regions were analyzed using Geneious software and the NCBI BLAST tool to assess the coverage and similarity of the gene sequences across all bacterial S. aureus strains. The results demonstrated that the conserved DNA region of the nuc gene, positions 210 – 373, served as the most reliable marker for detection. Primers and probes designed from this sequence exhibited high specificity and optimal performance in qPCR assays. These findings underscore the effectiveness of bioinformatics in selecting robust targets for qPCR-based diagnostic applications in food safety monitoring.