[Microplate hybridization methods of reduced unspecific reassociation considering delta Tm--comparison between relative relatedness of DNA and key characters for Mycobacterium szulgai].
It has been recognized that colorimetric microdilution plate hybridization method (DDH) shows equivocal identification results for some strains of Mycobacterium gordonae, and that chemotaxonomic identification method reveals some intermediate pattern between Mycobacterium szulgai and M. gordonae. In the present study, the results obtained by chemotaxonomic identification method on 25 strains of M. szulgai were compared with those obtained by DDH method. In chemotaxonomic methods, 8 of 25 M. szulgai strains showed negative results on 14 days' tween80 hydrolysis, 14 strains revealed negative nitrate reduction by the Tsukamura's method but all were positive by the Virtanen's method. Smooth colonies were found in 3 strains including M. szulgai type strain, JATA3201 (ATCC35799). Relative relatedness (relative color index) of genomic DNA was measured by DDH instead of spectrophotometric genomic DNA-DNA relatedness. A relative relatedness of 25 M. szulgai strains tested showed higher levels than 80%, but the inter-species relatedness to the other mycobacteria also showed high levels of 50-75%, when hybridizing temperature was set at 40 degrees C. At 56 degrees C, intra-species relative relatedness in 4 strains were lower than 50%, indicating that this condition is not appropriate. When hybridization temperature raised to 56 degrees C after overnight at 40 degrees C, a relative relatedness among 25 strains were again high (> 80%), and those to the other Mycobacterial species were lower than 70%. When hybridized at 56 degrees C after overnight at 45 degrees C, an intra-species relative relatedness again showed higher levels than 70% in all 25 strains, and interspecies percentiles were lowered satisfactorily to < 25%. In conclusion, through avoiding reassociation of nonspecific DNA fragments during the hybridization process, 45 degrees C overnight followed by 56 degrees C hybridization (delta DDH method) was found to be the better condition for identification and classification of Mycobacterium szulgai.
Fukasawa
Yutaka
Y
Department of Microbiology and Bioinformatics, Gifu University Graduate School of Medicine, 40, Tsukasa-machi, Gifu-shi, Gifu 500-8705, Japan. h2801105@guedu.cc.gifu-u.ac.jp
D004269:DNA, Bacterial / Q000032:analysis*; D009170:Nontuberculous Mycobacteria / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D013696:Temperature
D004269:DNA, Bacterial