Development loop-mediated isothermal amplification assay for detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) is a major etiological agent of persistent diarrhea in pediatric and adult population globally and it is a leading cause of infant mortality in developing countries. Bacterial culture and biochemical assays are insufficient for accurately identifying EPEC strains. In contrast, PCR serves as a major molecular diagnostic method for EPEC. However, the application of PCR is limited due to high cost, time taking and technical requirements.

In this study loop mediated isothermal amplification LAMP) was developed for rapid, specific, easy and resource-friendly detection of EPEC. The LAMP assay was developed by designing specific lamp primers targeting eae genes, in combination with stx1 and stx2 lamp primers to enable EPEC detection. The reaction mixture, which contained 2.5 µL of isothermal amplification buffer, 10 mM of MgSO4 solution, 1.4 mM of dNTPs, 1.8 mM of FIP and BIP, 0.4 mM of F3 and B3, 0.2 mM of LB, 8U of Bst polymerase, and 2.0 µL of the target DNA template, was incubated at 62 C0 for 60 minutes. The designed lamp assay's performance was assessed utilizing 60 bacterial trains that were isolated locally. The assay attained 100% efficiency (60/60), 100% sensitivity (10/10), and 100% specificity (50/50). Furthermore, both the positive and negative predictive scores were 100%. Up to 0.05 pg of DNA could be detected in each reaction using the developed LAMP test. The traditional PCR, on the other hand, showed a detection limit of 5 pg/reaction. Additionally, the developed LAMP assay was able to detect up to 7 x 102 cfu/g stool in a spiked stool sample. PCR, however, can identify up to 7 x 104 cfu/g of stool. Notably, compared to conventional PCR, the LAMP assay has a 100-fold greater sensitivity. The result obtained from LAMP and PCR tests showed a perfect agreement with a kappa value of 1 (k = 1).

The developed LAMP assay demonstrated promising performance for the detection of EPEC under controlled laboratory conditions, offering a rapid and straightforward alternative to conventional diagnostic methods. Clinical validation using patient stool sample and further studies with larger sample sets are needed to fully establish the assay's performance and support its broader clinical application.