DETECTION OF VIBRIO CHOLERAE O1 USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION

Background and Aim: Cholera is an acute gastrointestinal disease caused by Vibrio cholerae. V. cholera is a water and foodborne pathogen, that cholera outbreaks are causing serious damage to human health. There are many techniques for V. cholerae detection, these techniques including culture and molecular methods such as PCR, real-time PCR, multiplex PCR, NASBA, but these techniques are time-consuming and require expensive equipment. The aim of this work is detection of V. cholerae O1 using loop-mediated isothermal amplification. Methods: For this, Zot(Zonula occludens toxin) was selected for detection and specific primers were designed with utilization of Primer Explorer v4 software. The reaction mixture contains Bst buffer, dNTP, Betaine, FIP and BIP(inner primers), F3 and B3 (outer primers), Mgso4, template DNA. The reaction mixture during the initial denaturation at 94°C for 5 minutes in a heating block was placed, then the Bst DNA polymerase was added and the reaction mixture was incubated for 90 minutes at 65°C. Then LAMP products were analyzed with electrophoresis in2% agarose gel. Results: Agarose gel electrophoresis to examine the positive control of DNA amplification is a ladder-shaped smears and negative control samples genome lacks a primer dimer is observed. Conclusion: The LAMP method was providing a sensitive and rapid diagnostic assay for detection V. cholerae O1 without the need for expensive device.

Vibrio cholerae O1, LAMP, Detection, Zot gene