Optimization of the method for detecting stx₁, stx₂ and eae genes according to ISO 13136:2012 using endpoint PCR from E. coli strains
DOI:
https://doi.org/10.62035/rca.6.82Keywords:
foodborne diseases, enterohemorrhagic escherichia coli, escherichia coli O157, shiga-toxigenic escherichia coli, validation study, polymerase chain reactionAbstract
Shiga toxin-producing Escherichia coli (STEC) causes sporadic cases and outbreaks of diarrhea, with or without bloody stools, as well as hemolytic uremic syndrome (HUS). The O157:H7 serotype is endemic and the most prevalent in our country. Shiga toxins, intimin protein, and enterohemolysin are the main virulence factors. Foods at risk of STEC contamination include meat products, mainly those from butcher shops that do not implement good manufacturing practices or from unauthorized establishments. In this study, Annex C was optimized: Identification of STEC by multiple PCR amplification of virulence genes and detection of PCR products in agarose gel, of the ISO 13136:2012 “Microbiology of food and feedPCR-based methods for the detection of foodborne pathogens-Plaque method for the detection of STEC and the determination of serogroups O157, O111, O26, O103 and 0145”. This allowed for the implementation of a methodology to detect toxin-producing genes, stx₁, and stx₂, as well as the adherence factor gene eae. Additionally, the specificity and sensitivity of the methodology were verified, along with the evaluation of the robustness of the dye, thus guaranteeing the reliability of the results. The implementation of this methodology enables the Microbiology Service to confirm the STEC strains, isolated from food samples, using endpoint PCR, a more cost-effective technique than real-time PCR. This will help the laboratory optimize resources while facilitating the implementation of fundamental prevention and control strategies to reduce the morbidity and mortality associated with HUS and collaborate with the epidemiological studies in our country.
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