Application and evaluation of Loop Mediated Isothermal Amplification (LAMP) diagnostic assay for detection of Enterohemorrhagic Escherichia Coli (O157:H7)

Abstract

Abstract Backgrounds: O157:H7 E. coli has been implicated in the worldwide outbreak of food and water borne diseases. Early and effective diagnosis are the pivotal concerns for clinics and hospitals for prompt reporting of outbreaks, prevent more cases and treat the infection in appropriate time. Most of common diagnostic methods are constrained by significant drawbacks and novel reliable and rapid detection of this pathogen is beneficial to execute, particularly in low-resource laboratory settings. This study aimed at evaluating the performance of LAMP diagnostic assay targeting on putative fimbria protein coding gene (Z3276) for rapid and specific detection of EHEC O157:H7. Results: A total number of 40 locally available bacteria isolates and reference 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 included to optimization and evaluation for LAMP assay. The LAMP amplified DNA samples were visualized as turbid DNA with naked eye as well as using gel electrophoresis followed by staining. The assay performed with 100% (6/6) sensitivity, 97.05% (33/34) specificity, as well as 97.5% (39/40) efficiency. The assay was exhibited with 100% negative predicted value and 85.7% positive predicted value. The LAMP assay was also 10 times more sensitive than the conventional PCR assay; sensitivity evaluation was done through serial dilution. Additionally, LAMP and PCR assay results showed very high agreement (k = 0.97) on detection of the studied bacteria. Conclusions: Different diagnosis methods have been developed for EHEC O157:H7 detection. Nevertheless, some of the developed assays are not effective and others are expensive. In this study simple, rapid, specific and sensitive molecular diagnostic assay (LAMP assay) for detection of EHEC O157: H7 were evaluated. Thus, in comparison with performance of PCR and SMAC, LAMP assay was loftier in sensitivity, rapidness and cost-efficiency. Through further improvement, LAMP assay can be exploiting as point care diagnostic assays in resource-limited laboratories, especially in rural clinics and primary hospitals.