Identification of ISMyo2, a novel insertion sequence element of IS21 family and its diagnostic potential for detection of Mycobacterium yongonense

Background: Mycobacterium yongonense, as a novel member of the M. avium complex (MAC), was recently reported to be isolated from human specimens in South Korea and Italy. Due to its close relatedness to other MAC members, particularly M. intracellulare in taxonomic aspects, the development of a novel diagnostic method for its specific detection is necessary for clinical or epidemiologic purposes. Methods: Using the Mycobacterium yongonense genome information, we have identified a novel IS-element, ISMyo2. Targeting the ISMyo2 sequence, we developed a real-time PCR method and applied the technique to Mycobacterial genomic DNA. Results: To identify proper nucleic acid targets for the diagnosis, comparisons of all insertion sequence (IS) elements of 3 M. intracellulare and 3 M. yongonense strains, whose complete genome sequences we reported recently, led to the selection of a novel target gene, the M. yongonense-specific IS element, ISMyo2 (2,387 bp), belonging to the IS21 family. Next, we developed a real-time PCR method using SYBR green I for M. yongonense-specific detection targeting ISMyo2, producing a 338-bp amplicon. When this assay was applied to 28 Mycobacterium reference strains and 63 MAC clinical isolates, it produced amplicons in only the 6 M. yongonense strains, showing a sensitivity of 100 fg of genomic DNA, suggesting its feasibility as a diagnostic method for M. yongonense strains. Conclusions: We identified a novel ISMyo2 IS element belonging to the IS21 family specific to M. yongonense strains via genome analysis, and a real-time PCR method based on its sequences was developed.