Morbidity rate of tuberculosis and pulmonary disaeses causes caused by mycobacteria other than tubercle bacilli (MOTT) is very high throughout the world. The diagnosis of these diseases is primarily dependent on isolation of the pathogenic
mycobacteria
and subsequent identification by biochemical testing, Such procedures usually require 4-8 weeks. Identification of mycobacteria by this conventional method is time-consuming and laborious. In this study, mycobacterial species were differentiated
by
polymerase chain reaction(PCR)-restriction fragment length polymorphism (RFLP) analysis. Two kinds of primer sets were used for PCR amplification of mycobacterial DNA. One primer set(P1/P2) for identification of Mycobacterium tuberculosis was
based
on
the sequence of insertion sequence 6110, targetting DNA fragment of 123 basepars (bp) in size. The other primer set(D1/D2) for detetion of bacteria belonging to the genus Mycobacterium was based on the dnaJ sequence. The target DNA size of the
D1/D2
primer was 236bp.
D1/D2 PCR was performed using DNA isolated from standard mycobacterial strains. The 236bp amplified DNA was digested with 4 kinds of restriction endonucleases, NaeI, HinfI, FokI, or SmaI, and the RFLP pattern was analyzed, Six mycobacterial
species
could be differentiated by this method. The remaining 11 mycobacterial species was divided into 4 different groups. Among 83 isolated bacterial strains, 43 strains were found to be mycobacteria, which showed 236bp amplified DNA by D1/D2 PCR.
Tweenty five strains of mycobacteria were identified to be M. tuberculosis, which produced 123bp DNA bv P1/P2 PCR.
Eighteen clinically clinically isolated MOTT strains were analyzed by PCR-RFLP method. Nine strains were identified; 1 strain of M. scroflaceum, 3 strains of M. gordonae, 5 strains of M. szulgai. The remaining 9 strains were divided into 3
groups;
3
strains of M. kansasii-M. marinum-M. gastri group, 5 strains of M. avium-M. paratuberculosis group, and 1 strain of M. xenopi-M. fortuitum-M. chelonei group( This suggested that clinically isolated mycobacteria could be identified or, at least,
differentiated into several groups by RFLP analysis.
Specific DNA bands were amplified by both P1/P2 PCR and D1/D2 PCR using DNA extracted from clinical specimens. This showed that PCR-RFLP analysis could also be applied to the identification of mycobacteria in clinical specimens from patients with
pulmonary diseases.
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