| Abstrakt: |
Introduction: Enterohemorrhagic E. coli O157 infections are the leading cause of Hemolytic Uremic Syndrome (HUS) in children. Consumption of raw or undercooked beef from infected cattle can transmit the bacteria, which can adhere to human enterocytes and release Shiga toxins 1 and 2 (Stx 1 and 2), traveling through the bloodstream and severely affecting the kidneys and other smooth tissues. Objective: To compare the diagnostic capability of PCR techniques for detecting E. coli O157 in beef by amplifying the virulence genes of stx1 and stx2. Methods: DNA was extracted from E. coli O157:H7, ATCC 43895, and protocols for PCR and qPCR were developed to detect stx1 and stx2, validating each with cutoff limits, inclusivity, exclusivity, robustness, and testing on a meat matrix. Results and Discussion: Cutoff limits for PCR were 3.3x10-2 ng μL-1 and 9.9x10-2 ng μL-1 for stx1 and stx2, respectively; for qPCR, they were 1.7x10-3 ng μL-1 and 3.5x10-3 ng μL-1 for stx1 and stx2, respectively. Both techniques showed 100 % inclusivity, exclusivity, and robustness. In the artificially contaminated meat matrix, detection was achieved up to 4 CFU mL-1 for both qPCR and PCR, with the latter being less reliable. These results confirm the high sensitivity and specificity of molecular techniques, without the need for sample enrichment as required by culture tests, enabling rapid and reliable results to prevent potential foodborne infections. Conclusion: With the validation of PCR and qPCR protocols for stx1 and stx2, these could be implemented as rapid and reliable diagnostic techniques for detecting contamination in meat with a reduced number of E. coli O157, semi-quantitatively with PCR, or quantitatively with qPCR. [ABSTRACT FROM AUTHOR] |
