Korean J Clin Microbiol.  2011 Jun;14(2):41-47. 10.5145/KJCM.2011.14.2.41.

Characteristics of Molecular Strain Typing of Mycobacterium tuberculosis Isolated from Korea

Affiliations
  • 1Department of Laboratory Medicine, School of Medicine, Pusan National University, Busan, Korea.
  • 2Department of Thoracic and Cardiovascular Surgery, School of Medicine, Pusan National University, Busan, Korea. domini@pnu.edu

Abstract

Molecular strain typing of Mycobacterium tuberculosis is important for the detection of outbreaks of tuberculosis and laboratory cross contamination, as well as the differentiation between re-infection and reactivation of tuberculosis. In the present review, the authors investigated the currently available typing methods for M. tuberculosis and the current status of strain distribution in Korea. IS6110-restriction fragment length polymorphism (RFLP), which is considered a standard method, is based on numbers and positions of the insertion sequence, IS6110. The method has an excellent discriminatory power with a considerable amount of worldwide data, although it is time-consuming and labor-intensive. Spoligotyping is based on the presence or absence of spacer sequences between direct repeat (DR) regions. PCR amplification allows for the possibility of application in the early suspicious stage. The data can be easily digitized; however, it shows identical profiles in Beijing family strains. Mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) is another PCR-based genotyping method with a good discrimination power whose data can also be easily digitized. In Korea, the prevalence of Beijing family strains have been as high as 80 to 87%.

Keyword

Mycobacterium tuberculosis; Molecular strain typing; Beijing family

MeSH Terms

Discrimination (Psychology)
Disease Outbreaks
Humans
Korea
Mycobacterium
Mycobacterium tuberculosis
Polymerase Chain Reaction
Prevalence
Repetitive Sequences, Nucleic Acid
Sprains and Strains
Tandem Repeat Sequences
Tuberculosis

Figure

  • Fig. 1. The principles and procedures of IS6110-RFLP. (A) IS6110-RFLP for epidemiological investigation used the characteristics of polymorphism and copy number of IS6110. Extracted genomic DNA of M. tuberculosis was digested with a frequently cutting restriction enzyme, PvuII. IS6110 has only one restriction site for PvuII. Chromosomal regions other than IS6110 have randomly dispersed multiple restriction sites for PvuII. Therefore, lengths of fragments containing one end of IS6110 are variable; (B) DNA fragments under well-defined conditions were separated on agarose gel, and transferred to a nylon membrane; (C) hybridization with a fragment of the IS6110 sequence developed only IS6110-containing bands; (D) an image of real IS6110-RFLP patterns.

  • Fig. 2. The principles and procedures of DR spoligotyping. (A) Spoligotyping is based on the polymorphism in the direct repeat locus of the mycobacterial chromosome. The well-conserved 36-bp direct repeats are interspersed with unique spacer sequences varying from 35 to 41 bp in size. Deletion of some spacer regions between DRs is the main cause of strain diversity. Because the DRs have the same sequence, PCR generates fragments containing spacer sequences that are present in a specific strain; (B) Each PCR fragment contained at least one spacer sequence, although it may have many spacer sequences; (C) In a membrane, every probes targeting each spacer sequencer are spotted. PCR fragments are hybridized to the spots. The presence or absence of each spot generated unique spoligotyping pattern.

  • Fig. 3. The principles and procedures of MIRU-VNTR. (A) MIRU-VNTRs scattered throughout the whole bacterial genome contain variable copy numbers of repeat sequences. Each isolate is typed according to the number of copies of repeated units; (B) Sequences of variable number tandem repeats are amplified by PCR. The sizes of the amplified produces are dependent on the repeat copy numbers; (C) their sizes are determined by gel electrophoresis.


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