Visual Detection of Enterohemorrhagic Escherichia coli O157:H7 Using Loop-Mediated Isothermal Amplification

Mohsen Mohammadi, Lorestan University of Medical Sciences and Reza Ranjbar, Baqiyatallah University of Medical Sciences and Maryam Erfanmanesh, Zanjan University and Davoud Afshar, Zanjan University of Medical Sciences and Omar Ghaderi, University of Medical Sciences, Tehran, Iran and Ali Haghnazari, Zanjan University (2016) Visual Detection of Enterohemorrhagic Escherichia coli O157:H7 Using Loop-Mediated Isothermal Amplification. Electronic Physician. ISSN 2008-5842

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Abstract

Introduction: Escherichia coli O157:H7, an important foodborne pathogen, can cause serious renal damage, which can also lead to mortality. Since a rapid and sensitive method is needed to identify this pathogenic agent, we evaluated Loop-Mediated Isothermal Amplification Assay (LAMP) to detect Escherichia coli O157:H7. Methods: We used six primers that specifically identified the rfbE gene. To examine the sensitivity of the method, different dilutions were subjected to the LAMP reaction. Other bacterial strains also were investigated to determine the specificity of the test. The turbidity of the amplified products was assayed by visual detection. The amplified products were detected by addition of SYBR Green II to the reaction tubes. Results: Amplification products were observed as a ladder-like pattern on the agarose gel. A white turbidity emerged in the positive tubes. Under UV light, the positive samples were green, whereas the negative samples were orange. The detection limit of the LAMP was 78 pg/tube, and this indicated that it was 100 times more sensitive than PCR for the detection of EHEC. No LAMP products were detected when template DNA of non- EHEC strains were used, suggesting high specificity of the LAMP assay. Conclusion: The results indicated that the LAMP assay is a valuable diagnostic assay to identify EHEC O157:H7. In addition, the simplicity, sensitivity, specificity, and rapidity of this assay make it a useful method to diagnose pathogens in primary labs without any need for expensive equipment or specialized techniques. Keywords: Enterohemorrhagic Escherichia coli O157:H7, rfbE gene, Loop-Mediated Isothermal Amplification, SYBR Green II

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