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Jang, Choi, Chang, and Kim: Characteristics of Molecular Strain Typing of Mycobacterium tuberculosis Isolated from Korea
Original Article

Korean J Clin Microbiol 2011;14(2):41-47.

Published online: 21 June 2011

DOI: https://doi.org/10.5145/KJCM.2011.14.2.41

Characteristics of Molecular Strain Typing of Mycobacterium tuberculosis Isolated from Korea

Mi Hee Jang1, Go Eun Choi1, Chulhun L. Chang1, Yeong Dae Kim2

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

Correspondence: Yeong Dae Kim, Department of Thoracic and Cardiovascular Surgery, Pusan National University Hospital, 305 Gudeok-ro, Seo-gu, Busan 602-739, Korea. (Tel) 82-51-240-7822, (Fax) 82-51-243-9389, (E-mail) domini@pnu.edu.

Received 14 December 2010       Revised 7 January 2011       Accepted 7 January 2011

Copyright © 2011 Korean Journal of Clinical Microbiology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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%.

Keywords: Mycobacterium tuberculosis, Molecular strain typing, Beijing family

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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.
kjcm-14-41f1.tif
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.
kjcm-14-41f2.tif
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.
kjcm-14-41f3.tif
Table 1.
Characteristics of molecular strain typing methods fre quently used for M. tuberculosis
Methods Advantages Disadvantages
IS6110 RFLP Well standardized Reproducible Time consuming Laborious
    Difficult to discriminate low- or high-copy number isolates
Spoligotyping Rapid Single pattern in Beijing family
  Small sample required  
  Reproducible  
  Data digitized  
MIRU-VNTR Rapid Discrimination varies in different loci
  Small sample required  
  Reproducible  
  Data digitized  
  Economic  
SNP Rapid Requires efficient target selection and standardization
  Small sample required  
  Reproducible  
  Diverse within Beijing family  

Abbreviations: RFLP, restriction fragment length polymorphism MIRU-VNTR, mycobacterial interspersed repetitive unit-variable number of tandem repeat; SNP, single nucleotide polymorphism.

Table 2.
Frequency of Beijing family of M. tuberculosis in Korea
Sources of clinical isolates Periods No. of strains investigated Prevalence (%) of specificstrains References
KIT 1992∼1994 14 B, 43 [30]
KIT 1995∼1996 138 B, 72 [31]
One hospital in Taegu 1997∼1999 54 Not detected [35]
KIT 1998∼1999 206 K, 18 [4]
Public Health Centers in Gyeonggi Province 2004 715 K, 24 [36]
One tuberculosis hospital 2005∼2006 208 B, 97 [34]
Nationwide 2006 80 B, 80 [32]
Two groups combined (Onehospitalin Busan, and KIT) 2007 187 B, 22 [25]
11 university hospitals 2008∼2009 96 B, 87; K, 15 [33]

Abbreviations: KIT, Korean Institute of Tuberculosis; B, Beijing family; K, K strain.

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