Establishment of loop-mediated isothermal amplification for rapid and convenient detection of Mycobacterium marinum...

Mycobacterium marinum is a zoonotic pathogen that can cause dermatological infection mainly from contaminated water or fish. Some well-known genetically similar species and subspecies are M. lifrandii and M. pseudoshottsii from amphibians and fish in aquaculture, and M. ulcerans, a causative agent of a neglected tropical disease (NTD), Buruli ulcer. They are believed to survive in water as their major niche, which might be related to their source of infection, but detailed ecological surveillance of the species complex remains to be done. Herein, we present a new detection system for M. marinum complex based on isothermal DNA amplification that can be conducted conveniently with high sensitivity and specificity. The target was a chromosomal gene, mrsA, including a restriction polymorphism between M. ulcerans (except for the most ancestral subspecies, M. ulcerans subsp. shinshuense) and the other species. The system was able to detect less than 500 fg (approximately 70 copies) of genomic DNA of M. marinum, within 60 min, and caused no amplification from mycobacterial species other than M. marinum complex species. It was also verified that restriction of the amplified DNA fragments was able to discriminate M. ulcerans as expected. This easy, quick, and convenient system is expected to facilitate detection of M. marinum complex from various resources.