Yonsei Med J.  2013 Sep;54(5):1220-1226. 10.3349/ymj.2013.54.5.1220.

A Simple and Efficient Multiplex PCR Assay for the Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex to the Species Level

• Kim Y
• Choi Y
• Jeon BY
• Jin H
• Cho SN
• Lee H

• KMID: 1793171
• DOI: http://doi.org/10.3349/ymj.2013.54.5.1220

Abstract

PURPOSE
The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guerin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health.
MATERIALS AND METHODS
A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates.
RESULTS
All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level.
CONCLUSION
The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.

Keyword

M. tuberculosis complex; multiplex PCR; rpoB; RD1; RD8

MeSH Terms

Animals
Cattle
Classification/methods
DNA Primers
Genes, Bacterial
Humans
Multiplex Polymerase Chain Reaction/*methods
Mycobacterium/classification/genetics/*isolation & purification
Mycobacterium tuberculosis/classification/genetics/*isolation & purification
Species Specificity
DNA Primers

Figure

• 

  Fig. 1 Schematic diagram showing regions flanking the direct repeat region in M. tuberculosis H37Rv, M. bovis and M. bovis bacillus Calmette-Guérin (BCG). This region is specifically deleted in M. bovis and M. bovis BCG, indicated by dotted lines. Primer positions are indicated by small arrows. RD, regions of difference.

• 

  Fig. 2 Multiplex PCR for M. tuberculosis, M. bovis and M. bovis bacillus Calmette-Guérin (BCG) strains. PCR products were separated by electrophoresis and visualized. Lanes M: molecular size marker (100-bp ladder); 1: M. bovis BCG Pasteur; 2: M. bovis BCG Tice; 3: M. bovis BCG Tokyo; 4: M. bovis AN5; 5: M. tuberculosis H37Rv; 6: negative control. Expected sizes of PCR products are shown. RD, regions of difference; PCR, polymerase chain reaction; ESAT-6, early secreted antigenic target 6 kDa protein.

• 

  Fig. 3 Multiplex PCR for the Mycobacterium species. PCR products were separated by electrophoresis and visualized. Lanes M: molecular size marker (100-bp ladder); 1: M. avium; 2: M. africanum; 3: M. abscesses; 4: M. celatum; 5: M. chelonae; 6: M. fortuitum; 7: M. gordonae; 8: M. intracellulare; 9: M. kansasii; 10: M. marinum; 11: M. microtii; 12: M. phlei; 13: M. septicum; 14: M. szulgai; 15: M. terrae; 16: M. ulcerans; PCR, polymerase chain reaction.

• 

  Fig. 4 Multiplex PCR for clinical M. tuberculosis isolates. PCR products were separated by electrophoresis and visualized. Lanes M: molecular size marker (100-bp ladder); 1-15: M. tuberculosis isolates; 16: M. tuberculosis H37Rv; 17: M. tuberculosis Erdman; PCR, polymerase chain reaction.

• 

  Fig. 5 Multiplex PCR for clinical M. bovis isolates. PCR products were separated by electrophoresis and visualized. Lanes M: molecular size marker (100-bp ladder); 1-20: M. bovis isolates; 21: M. bovis AN5; 22: M. bovis BCG Pasteur; 23: M. tuberculosis H37Rv. BCG, bacillus Calmette-Guérin; PCR, polymerase chain reaction.

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