Insertion sequence 6110의 mixed-linker 중합효소 연쇄반응을 이용한 M.tuberculsosis의 molecualr

Abstract

Background Various classic typing methods including phage typing, antibiotic resistance typing and multilocus enzyme electrophoresis have been proven inefficient because of the phenotypic and genotypic homogeneities of the Mycobacterium tuberculosis(M.tuberculosis) species. Thus, DNA restriction fragment length polymorphism(RFLP) analysis using the repetitive element insertion sequence 6110(IS6110) of the M. tuberculosis genome has been proposed to be a powerful epidemiologic tool. However, this method requires large amount of genomic DNA and Southern hybridization procedures. The aim of this study was to evaluate DNA RFLP analysis by mixed-linker polymerase chain reaction(mixed-linker PCR) for a rapid, simple typing method, which specifically amplified genomic RFLP fragments containing IS6110 with using one primer specific for IS6110 and a second primer complementary to a linker ligated to the restricted genomic DNA.

Methods Fourteen clinical isolates of M. tuberculosis with various drug susceptibilities including 2 strains isolated from a patient were used for the study. Their RFLP patterns by mixed linker-PCR(mixed liker RFLP) were analyzed and compard with those by the conventional RFLP method.

Results Mixed-linker RFLP obtained from 13 isolates of 13 patients showed all different reproducible patterns and 2 isolates from the same patient showed almost the same RFLP patterns which had different drug susceptibility. mixed-linker RFLP patterns were correlated to the results by the conventional RFLP. And mixed-linker RFLP showed a higher number of fragments containing IS6110 than those by the conventional RFLP. However, both mixed-linker and conventional RFLP were independent of drug resistance patterns.

Conclusion Mixed linker-RFLP is a rapid, simple typing method with high discrimination and reproducibility, and seems to be useful for epidemiologic study of M. tuberculosis.