J Korean Med Sci.  2016 Nov;31(11):1673-1683. 10.3346/jkms.2016.31.11.1673.

Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods

• Ei PW
• Aung WW
• Lee JS
• Choi GE
• Chang CL

• KMID: 2470260
• DOI: http://doi.org/10.3346/jkms.2016.31.11.1673

Abstract

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units-variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.

Keyword

Tuberculosis; Mycobacterium tuberculosis; Molecular Strain Typing

MeSH Terms

DNA Transposable Elements/genetics
High-Throughput Nucleotide Sequencing
Humans
Molecular Typing/*methods
Mycobacterium tuberculosis/genetics/*isolation & purification/metabolism
Nucleic Acid Hybridization
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
Tandem Repeat Sequences/genetics
Tuberculosis/microbiology
DNA Transposable Elements

Figure

• 

  Fig. 1 Principles of RFLP. (A) M. tuberculosis genome with insertion segment IS6110 showing the PvuII cleavage sites. (B) Digestion of the whole genome with PvuII. (C) DNA segments of different sizes after run in the gel are transferred onto a membrane followed by hybridization. (D) Visualized fragments, which represent a single copy of IS6110 surrounded by flanking DNA of different lengths.

• 

  Fig. 2 Principle of spoligotyping and the processing of signals. (A) M. tuberculosis genome with well-conserved 36-bp direct repeats (DRs) which are interspersed by 35–43 bp of unique spacer sequences. Genetic diversity depends on the deletion of these spacer regions. The spacer regions are amplified by primers, and the presence of at least one spacer fragment shows a PCR positive reaction. On the membrane, 43 probes targeting each spacer are spotted, and a unique pattern of spoligotyping is visualized after hybridization with PCR product. (B) Signals of reference strain H37Rv. (C) A typical signal pattern of Beijing family M. tuberculosis strain. (D) To analyze signal patterns, the signals are converted to binary code of ‘on (1) and off (0)’. (E) The 43-digit binary code is converted to a 15-digit octal (i.e., base 8, having the digits 0–7) designation by a two-step process. First, the 43-digit binary code is divided into 14 sets of three digits (spacers 1 through 42) plus one additional digit (spacer 43). (F) Each 3-digit binary set is converted to its octal equivalent, with the final additional digit remaining as 1 or 0. The translation of binary numbers to octal numbers is done as follows: 000 = 0; 001 = 1; 010 = 2; 011 = 3; 100 = 4; 101 = 5; 110 = 6; and 111 = 7.

• 

  Fig. 3 Principle of MIRU-VNTR genotyping. (A) MIRU-VNTR loci of different repetitive numbers scattered in M. tuberculosis genome are amplify by specific primers for each locus. (B) Different sizes of amplicons after PCR. (C) Amplicons can be seen after gel electrophoresis with different sizes that reflect the repetitive number of each VNTR locus.

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