Development of Loop Mediated Isothermal Amplification for Rapid and Sensitive Detection of Enteroaggregative Escherichia coli (EAEC)

Abstract Enteroaggregative Escherichia coli (EAEC) is a significant etiologic agent for acute and persistent diarrhea in children and adults globally. Bacterial culture and biochemical assays are insufficient for accurately identifying EAEC strains. Currently, the HEp-2 cell assay is the gold standard method for the detection of EAEC. However, its use is restricted to the reference laboratories due to the technical complexity and infrastructure requirement. In contrast, PCR has been used as a major molecular method for detecting EAEC strains; However, its routine use in diagnostic laboratories is limited by several factors. In this study loop mediated isothermal amplification (LAMP) was developed for affordable, rapid and specific detection of EAEC. The assay was developed by using a specifically designed lamp primer targeting aaic genes to enable precise detection of EAEC. The performance of the developed assay was evaluated by using 60 locally isolated bacterial strains. The assay exhibited 100% sensitivity and a specificity. 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 detection limit of 80 CFU/mL, which is higher than 8× 10 2 CFU/mL for conventional PCR, indicating the LAMP assay’s 10-fold higher sensitivity than conventional PCR. Furthermore, in spiked stool sample the lowest detection limit for LAMP was 8 × 10 2 cfu/ g stool. In contrast, 8 × 10 4 cfu/g stool for PCR. Notably, the LAMP assay shows 100-fold higher sensitivity than conventional PCR.