Specific and Rapid Detection of Zonula Occludens Toxin-producing Vibrio Cholerae Using LAMP

Vibrio cholerae is one of the most important infectious human pathogens among the toxicogenic strains during the history of many pandemics and is a global threat to public health, especially in developing countries. Therefore, rapid and on-time detection of this infectious pathogen is necessary to prevent its outbreak. The aim of this work was the identification of zonula occludens toxin produced by V. cholerae using the LAMP technique. In this study, zot (zonula occludens toxin), one of the virulence factors of V. cholerae, was selected as the target gene and specific primers for the sequence of this gene were designed using the PrimerExplorer V5 software. The optimization of various factors affecting the LAMP reaction including Mg2+ ion, primers, temperature, and incubation time was performed in a traditional way and also by Taguchi test design. Finally, the LAMP products were visualized by agarose gel electrophoresis stained with ethidium bromide and SYBR Green I fluorescent dye. The data showed that the optimum condition for the LAMP reaction was 4-12 mM Mg2+ ion, 1.6-0.8 and 0.53 μM FIP/BIP, 0.4-0.2 and 0.13 μM F3/B3, temperature of 60-65 °C, and incubation time of 30- 90 min. However, using Taguchi method, the optimum condition was 6 mM MgSO4, incubation time of 60 min, and temperature of 65 °C. In conclusion, the results of this study showed that the LAMP method provides the rapid, sensitive, and specific detection of zonula occludens toxin-producing V. cholerae and can be used for the design of an identification kit of this pathogen.