Abstract
Shiga toxin-producing Escherichia coli (STEC) is a group of human foodborne pathogens transmitted to humans through the consumption of different types of food. Their detection is mainly performed by targeting specific serogroups by classical microbiological methods and, later, by molecular typing with different techniques. The application of multiplex real-time PCR (qPCR) can significantly improve the turnaround time of the existing methodologies as in one single run it is possible to detect and characterize specific microorganisms. In the present chapter, a pentaplex qPCR assay is described for the identification of STEC which may also be applied for the rapid screening of these pathogens in different types of foods. The assay targets the most important virulence factors of these microorganisms, the genes stx1, stx2, and eae, along with the rfbE gene which encodes for the “O157” antigen as this is the most prevalent serogroup among all STEC, as well as an internal amplification control to rule out false-negative results due to qPCR inhibition.
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References
Kim SO, Kim SS (2021) Bacterial pathogen detection by conventional culture-based and recent alternative (Polymerase Chain Reaction, isothermal amplification, Enzyme Linked Immunosorbent Assay, bacteriophage amplification, and gold nanoparticle aggregation) methods in food samp. J Food Saf 41:1–12
ISO/TS (2012) Microbiology of food and animal feed – Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens – horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157
Feng P, Weagant SD, Jinneman K (2011) Diarrheagenic Escherichia coli. http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070080.htm
Garrido-Maestu A, Chapela M-J, Peñaranda E et al (2015) Re-evaluation of enhanced qPCR prevalidated method for next-day detection of Salmonella spp., Shigella spp., Escherichia coli O157 and Listeria monocytogenes. Food Biotechnol 29:317–335
Bundidamorn D, Supawasit W, Trevanich S (2021) Taqman® probe based multiplex RT-PCR for simultaneous detection of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli in foods. LWT 147:111696
Villamizar-Rodríguez G, Lombó F (2017) Multiplex Detection of Food-Borne Pathogens. In: Domingues L (ed) PCR: methods and protocols. Springer New York, New York, pp 153–162
Dhital R (2021) Detection of virulence and extended spectrum β-lactamase genes in Salmonella by multiplex high-resolution melt curve real-time PCR assay. J Appl Microbiol 132(3):2355–2367
Müştak İB, Müştak HK (2022) Detection and differentiation of Salmonella Enteritidis and Salmonella Typhimurium by multiplex quantitative PCR from different poultry matrices. Br Poult Sci 63:171–178
Bundidamorn D, Supawasit W, Trevanich S (2018) A new single-tube platform of melting temperature curve analysis based on multiplex real-time PCR using EvaGreen for simultaneous screening detection of Shiga toxin-producing Escherichia coli, Salmonella spp. and Listeria monocytogenes in food. Food Control 94:195–204
Vitullo M, Grant KA, Sammarco ML et al (2013) Real-time PCRs assay for serogrouping Listeria monocytogenes and differentiation from other Listeria spp. Mol Cell Probes 27:68–70
Raymaekers M, Smets R, Maes B et al (2009) Checklist for optimization and validation of real-time PCR assays. J Clin Lab Anal 23:145–151
Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Deer DM, Lampel KA, González-Escalona N (2010) A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays. Lett Appl Microbiol 50:366–372
Hoorfar J, Malorny B, Abdulmawjood A et al (2004) Practical considerations in design of internal amplification controls for diagnostic PCR assays MINIREVIEW practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol 42:1863–1868
Garrido-Maestu A, Azinheiro S, Carvalho J et al (2019) Combination of immunomagnetic separation and real-time recombinase polymerase amplification (IMS-qRPA) for specific detection of Listeria monocytogenes in smoked salmon samples. J Food Sci 84(7):1881–1887
Garrido-Maestu A, Azinheiro S, Carvalho J et al (2018) Development and evaluation of loop-mediated isothermal amplification, and Recombinase Polymerase Amplification methodologies, for the detection of Listeria monocytogenes in ready-to-eat food samples. Food Control 86:27–34
Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3-new capabilities and interfaces. Nucleic Acids Res 40:e115–e115
Mackay IM (2004) Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 10:190–212
Biassoni R, Raso A (eds) (2020) Quantitative real-time PCR methods and protocols. Humana, New York, NY
CLC Bio-Qiagen (2016) CLC Sequence Viewer
Bikandi J, San Millán R, Rementeria A et al (2004) In silico analysis of complete bacterial genomes: PCR, AFLP–PCR and endonuclease restriction. Bioinformatics 20:798–799
Kralik P, Ricchi M (2017) A basic guide to real time PCR in microbial diagnostics: definitions, parameters, and everything. Front Microbiol 8:1–9
Costa-Ribeiro A, Azinheiro S, Fernandes SP, Lamas A, Prado M, Salonen LM, Garrido-Maestu A (2023) Evaluation of covalent organic frameworks for the low-cost, rapid detection of Shiga Toxin-producing Escherichia coli in ready-to-eat salads. Anal Chim Acta 341357. https://doi.org/10.1016/j.aca.2023.341357
Garrido-Maestu A, Azinheiro S, Carvalho J et al (2020) Optimized sample treatment, combined with real-time PCR, for same-day detection of E. coli O157 in ground beef and leafy greens. Food Control 108:106790
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Costa-Ribeiro, A., Azinheiro, S., Roumani, F., Prado, M., Lamas, A., Garrido-Maestu, A. (2023). Multiplex Real-Time PCR for the Detection of Shiga Toxin-Producing Escherichia coli in Foods. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 2967. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3358-8_6
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DOI: https://doi.org/10.1007/978-1-0716-3358-8_6
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