Development of Loop-Mediated Isothermal Amplification Assay for the Detection of aaic Positive Enteroaggregative Escherichia coli (EAEC)

Abstract

Enteroaggregative Escherichia coli is a significant etiologic agent for diarrheal disease globally. Polymerase Chain Reaction (PCR) used as a major detection method for EAEC strains. However, its routine use in diagnostic laboratories is limited by several factors. In this study, a Loop-Mediated Isothermal Amplification (LAMP) assay was developed as an alternative molecular method for detecting aaic positive EAEC strains. The LAMP reaction was performed by incubating the reaction components including 4 sets of designed primers, isothermal buffer, MgSO4, Bst polymerase, dNTPs, and template DNA in a water bath at 61°c for 1 h. The assay performance was evaluated by using 60 bacterial strains and showed high analytical sensitivity, specificity and efficiency on limited strain panel. The developed LAMP assay could detect up to 0.098pg of DNA/reaction. In contrast, the conventional PCR exhibited a detection limit of 0.98pg/reaction. In pure culture, the developed LAMP assay has a lower detection limit of 80 CFU/mL, which is 10-fold lower than that of conventional PCR (8 × 10² CFU/mL), indicating its higher sensitivity. Additionally, in a spiked feces sample, the lowest detection limit for LAMP was 8.2 × 102 cfu/g stool, but the lowest detection limit for PCR was 8.2 × 104 cfu/g stool. Notably, the LAMP assay shows 100-fold lower detection than conventional PCR. The developed LAMP assay detects aaic-positive strains and forms a promising foundation for future multiplex assay development aimed at comprehensive EAEC detection.