Development of a SYBR Green qPCR Intralaboratory Validation for the Quantification of <i>Escherichia coli</i> O157:H7

Abstract

Escherichia coli serotype O157:H7 is a diarrheal agent and a significant cause of hemorrhagic colitis and the development of hemolytic uremic syndrome (HUS), mainly in infants. Early detection of contaminated food and water using reliable and fast tests is one of the strategies to prevent infections from E. coli O157:H7. Methods: Four quantitative polymerase chain reaction protocols (SYBR Green qPCR) were developed and validated to determine the presence of the bacteria according to its rfbE, stx1, and stx2 genes. Results: The results of the efficiencies were between 80% and 97% with a high linearity (R2 0.99). The cut-off limits for each primer sequence were 3.1667 × 10−2 ng µL−1 for two sequences of the serogroup O157 (primers rfbE and O157), 1.7228 × 10−3 ng µL−1 for stx1, and 3.5185 × 10−3 ng µL−1 for stx2. The inclusivity and the exclusivity of each gene, as well as the analytical precision and the positive and negative predictive value, were 100%. A contaminated meat matrix was evaluated, detecting up to 4 CFU g−1. Conclusions: SYBR Green qPCR protocols could be implemented to trace the presence of E. coli O157 in a routine analysis of ground beef or as an easy, rapid, sensitive, and specific diagnostic test while still considering microbiological tests to validate any inconclusive results.

Keywords

• SYBR Green
• qPCR
• <i>stx</i>₁
• <i>stx</i>₂
• <i>rfbE</i>
• validation