Loop mediated isothermal amplification as a molecular diagnostic assay: Application and evaluation for detection of Enterohaemorrhagic Escherichia coli (O157:H7).

Purpose: This study aimed at evaluating the performance of the Loop Mediated Isothermal Amplification (LAMP) diagnostic test, which targets the putative Fimbria protein-encoding gene (Z3276) for rapid and specific detection of locally isolated enterohemorrhagic Escherichia coli (EHEC) O157:H7. Results: A total number of 40 locally available bacteria isolates and standard strains, among them 6 entrohemorrhagic (O157:H7) and 10 entropathogenic E. coli, 7 non diarrheic E. coli strains and 13 non entrohemorrhagic shiga toxic (stx) E. coli isolates as well as 4 pathogenic non E. coli species were used to optimize and evaluate the LAMP assay. The LAMP amplified DNA samples were visualized as turbid DNA both by naked eye and gel electrophoresis followed by staining. The assay had a sensitivity of 100% (6/6), a specificity of 97.05% (33/34), and an efficiency of 97.5% (39/40). The assay was also exhibited with 100% negative predicted value and 85.7% positive predicted value. The LAMP assay was also 10-fold more sensitive than the conventional PCR assay; sensitivity was determined by serial dilution. The results of LAMP and the PCR tests showed very high agreement (k = 0.97) in the detection of the bacteria studied. Conclusion: Compared with the performance of PCR and SMAC, LAMP assay was better in terms of efficiency, rapidity and cost-effectiveness, which can be used as a point-care diagnostic test in resource-limited laboratories.

Designing of immunodiagnostic assay using polyclonal antibodies for detection of Shiga toxin producing pathogenic E. coli (STEC) strains.

Shiga toxin-producing Escherichia coli (STEC) are the leading causes of diarrhea and death of humans worldwide. Many diagnostic assays have been developed to aid for the diagnosis of STEC strain; however, they have limitations. Thus, this study was aimed at designing rapid, effective, sensitive and specific immunodiagnostic assay for STEC strain detection. Thus, a STEC isolate from Ethiopia was processed for LPS extraction and the LPS was used to immunize mice.. The produced antibody showed positive agglutination both on the purified LPS as well as the STEC isolate carrying LPS on their surface; however, agglutination of STEC was more pronounced. Mice immunized with LPS produced highest agglutination on tertiary immunization showing the progressive buildup of the antibody response against the antigen. Cultures from tryptone soya agar and when they refresh showed better agglutination than cultures on EMB as well as tryptone soya broth. Immunodiagnostic assay developed in this study could detect STEC strains including STEC in human feces rapidly (1-2 min), with high sensitivity (90.2%), specificity (89.5%) and accuracy (90.6%). However, further studies are still required to improve the sensitivity, specificity and reproducibility. Overall this diagnostic assay provided promising results that may curb current problem with detection methods in clinical health care and research laboratories.