Detection of Vibrio shilonii by using Loop-mediated isothermal amplification (LAMP)

Abstract:

[Background] Coral bleaching events occur frequently which causes the coral reef undergoing severe degeneration in recent years. Disease caused by pathogens is one of the reasons for coral bleaching. Vibrio shilonii was identified to be a pathogen that is able to infect the coral Oculina patagonica and cause its bleaching. However, its epidermology remains largely unknown due to the lack of a rapid diagnostic tool. [Objective] To establish a rapid and simple method for detection of V. shilonii using Loop-mediated isothermal amplification (LAMP) technique. [Methods] A set of 6 specific primers based on the target of rpoD sequence was designed for LAMP amplification. The sensitivity and specification of the primers were tested and conditions for LAMP amplification were optimized. The products of LAMP were visualized with fluorescent reagent of calcein and agarose gel electrophoresis respectively. The limitation of the method was compared with conventional PCR and Real-time PCR. [Results] The result showed that this method can specifically detect V. shilonii without showing cross-reaction with closely related vibrio species or other bacteria. The detection limit is visualized with calcein was 3.641×103 cps/mL of the plasmids carrying rpoD, which was 1 000 times more sensitive than conventional PCR and similar to that of Real-time PCR method; Meanwhile, this method was shown to be able to directly detect the pathogen in the seawater with a minimum concentration of 1.3×102 cfu/mL. [Conclusion] Overall, this study revealed that the method we developed has high specificity and sensitivity for diagnosis of V. shilonii. It is more sensitive and more convenient than the conventional PCR or real-time PCR. Therefore, it has the potential to be used for diagnosis in the field.