Harnessing bioinformatics to identify unique gene sequences tailored for the specific detection of Staphylococcus aureus by real-time PCR
7 viewsDOI:
https://doi.org/10.54939/1859-1043.j.mst.109.2026.95-103Keywords:
Bioinformatics; Real-time PCR; S. aureus; Nuc.Abstract
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.
References
[1]. Upadhyay N., Nara S., “Lateral flow assay for rapid detection of Staphylococcus aureus enterotoxin A in milk”, Microchem J, vol. 137, pp. 435–442, (2018). DOI: https://doi.org/10.1016/j.microc.2017.12.011
[2]. Cohen M. L., “Changing patterns of infectious disease”, Nature, vol. 406, no. 6797, pp. 762–767, (2000). DOI: https://doi.org/10.1038/35021206
[3]. Murray R. J., “Recognition and management of Staphylococcus aureus toxin-mediated disease”, Intern Med J, vol. 35, suppl. 2, pp. S106–S119, (2005). DOI: https://doi.org/10.1111/j.1444-0903.2005.00984.x
[4]. Scharff R. L., “Economic burden from health losses due to foodborne illness in the United States”, J Food Prot, vol. 75, no. 1, pp. 123–131, (2012). DOI: https://doi.org/10.4315/0362-028X.JFP-11-058
[5]. Schmid D., Fretz R., Winter P., et al., “Outbreak of Staphylococcal food intoxication after consumption of pasteurized milk products, June 2007, Austria”, Wien Klin Wochenschr, vol. 121, no. 3–4, pp. 125–131, (2009). DOI: https://doi.org/10.1007/s00508-008-1132-0
[6]. Quyen D. V. et al., “Isolation and phylogenetic analysis of Staphylococcus aureus strains isolated from meat in traditional markets in Ha Noi”, Acad J Biol, vol. 47, no. 1, pp. 63–74, (2025). DOI: https://doi.org/10.15625/2615-9023/21635
[7]. “Fish and fishery products hazards and controls guidance. Chapter 15: Staphylococcus aureus toxin formation in hydrated batter mixes”.
[8]. Lindsay J. A., Holden M. T. G., “Staphylococcus aureus: superbug, super genome?”, Trends Microbiol, vol. 12, no. 8, pp. 378–385, (2004). DOI: https://doi.org/10.1016/j.tim.2004.06.004
[9]. Yang Y., Su X., Yuan Y., et al., “Detection of Staphylococcus aureus in dairy products by polymerase chain reaction assay”, Agric Sci China, vol. 6, no. 7, pp. 857–862, (2007). DOI: https://doi.org/10.1016/S1671-2927(07)60122-9
[10]. Kobayashi N. et al., “Detection of mecA, femA, and femB genes in clinical strains of staphylococci using polymerase chain reaction”, Epidemiol Infect, vol. 113, no. 2, pp. 259–266, (1994). DOI: https://doi.org/10.1017/S0950268800051682
[11]. Martineau F., Picard F. J., Roy P. H., et al., “Species-specific and ubiquitous-DNA-based assays for rapid identification of Staphylococcus aureus”, J Clin Microbiol, vol. 36, no. 3, pp. 618–623, (1998). DOI: https://doi.org/10.1128/JCM.36.3.618-623.1998
[12]. Ma K., Deng Y., Bai Y., et al., “Rapid and simultaneous detection of Salmonella, Shigella, and Staphylococcus aureus in fresh pork using a multiplex real-time PCR assay based on immunomagnetic separation”, Food Control, vol. 42, pp. 87–93, (2014). DOI: https://doi.org/10.1016/j.foodcont.2014.01.042
[13]. McAdow M., Missiakas D. M., Schneewind O., “Staphylococcus aureus secretes coagulase and von Willebrand factor binding protein to modify the coagulation cascade and establish host infections”, J Innate Immun, vol. 4, no. 2, pp. 141–148, (2012). DOI: https://doi.org/10.1159/000333447
[14]. Khan A. et al., “Molecular analysis of virulent genes (coa and spa) of Staphylococcus aureus involved in natural cases of bovine mastitis”, Pak J Agric Sci, vol. 50, no. 4, pp. 739–743, (2013).
[15]. Wu S., Huang J., Wu Q., et al., “Prevalence and characterization of Staphylococcus aureus isolated from retail vegetables in China”, Front Microbiol, vol. 9, p. 1263, (2018). DOI: https://doi.org/10.3389/fmicb.2018.01263
[16]. Paterson G. K. et al., “Prevalence and characterization of human mecC methicillin-resistant Staphylococcus aureus isolates in England”, J Antimicrob Chemother, vol. 69, no. 4, pp. 907–910, (2014). DOI: https://doi.org/10.1093/jac/dkt462
[17]. Eita M. et al., “Large outbreak of Staphylococcus aureus and Bacillus cereus caused by ready-to-eat meals in Aomori, Japan”, Open Forum Infect Dis, vol. 12, suppl. 1, ofae631.1494, (2025). DOI: https://doi.org/10.1093/ofid/ofae631.1494
[18]. Montelongo C. et al., “Whole-genome sequencing of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates from Egypt”, Microbiol Spectr, vol. 10, no. 4, e02413-21, (2022). DOI: https://doi.org/10.1128/spectrum.02413-21
[19]. Wielders C. L. C., Fluit A. C., Brisse S., et al., “mecA gene is widely disseminated in Staphylococcus aureus population”, J Clin Microbiol, vol. 40, no. 11, pp. 3970–3975, (2002). DOI: https://doi.org/10.1128/JCM.40.11.3970-3975.2002
[20]. Ciesielczuk H. et al., “Methicillin-resistant Staphylococcus aureus harboring mecC still eludes us in East London, United Kingdom”, J Clin Microbiol, vol. 57, no. 6, (2019). DOI: https://doi.org/10.1128/JCM.00020-19
