Determination of DNA-DNA Hybridization Condition for Rapid Identification of Mycobacterium Species.
- Author:
Yun Sop CHONG
;
Sang Nae CHO
;
Kyung Won LEE
;
Hong Seok PARK
- Publication Type:Original Article
- MeSH:
Dextran Sulfate;
DNA;
Edetic Acid;
Heating;
Hot Temperature;
Humans;
Hydrogen Peroxide;
Methanol;
Mycobacterium*;
Peroxidase;
Salmon;
Sodium
- From:Journal of the Korean Society for Microbiology
1999;34(2):137-145
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
Rapid identification of Mycobacterium spp. isolated from patients is important with increased isolation of mycobacteria other than tubercle bacilli (MOTT). DNA-DNA hybridization with streptavidin-peroxidase and tetramethylbenzidine (TMB) color reaction method was recognized as a useful tool for identification of various species of mycobacteria. In this study, optimum condition of the test was determined. The optimal concentrations of tetramethylbenzidine dihydrochloride and hydrogen peroxide for streptavidin-horseradish peroxidase were 0.3-0.6 ug/ ml and 0.16 mM, respectively. The TMB stock solution was stable when prepared in methanol and the dilution of TBM stock solution in 10 mM sodium citrate-10 mM EDTA solution (pH 5.0) gave highest peroxidase-TMB activity. The suitable composition of hybridization solution consisted of 2 x SSC, 10% dextran sulfate, 50 ug/ml salmon DNA, 5 x Denhardt's solution, and 50% formamide. The 5-minute heating at 100C of test DNA prior to photobiotin labeling significantly increased the reaction. In conclusion, DNA-DNA hybridization method with streptavidin-peroxidase and TMB color reaction method may be useful for rapid identification of Mycobacterium spp. isolated from patients.
