Identification of a new DNA region specific for members of Mycobacterium tuberculosis complex

Abstract

The successful use of DNA amplification for the detection of tuberculous mycobacteria crucially depends on the choice of the target sequence, which ideally should be present in all tuberculous mycobacteria and absent from all other bacteria. In the present study we developed a PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system named SenX3-RegX3. The senX3-regX3 IR is composed of a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). In a survey of 116 Mycobacterium tuberculosis strains characterized by different IS6110 restriction fragment length polymorphisms, 2 Mycobacterium africanum strains, 3 Mycobacterium bovis strains (including 2 BCG strains), and 1 Mycobacterium microti strain, a specific PCR fragment was amplified in all cases. This collection included M. tuberculosis strains that lack IS6110 or mtp40, two target sequences that have previously been used for the detection of M. tuberculosis. No PCR fragment was amplified when DNA from other organisms was used, giving a sensitivity of 100% and a specificity of 100% in the confidence limit of this study. The numbers of MIRUs were found to vary among strains, resulting in six different groups of strains on the basis of the size of the amplified PCR fragment. However, the vast majority of the strains (approximately 90%) fell within the same group, containing two 77-bp MIRUs followed by one 53-bp MIRU.

FIG. 1

senX3-regX3 operon and PCR amplification strategy. The 3.2-kb EcoRI-BamHI fragment containing the senX3-regX3 operon from BCG 1173P2 is shown on the top. The black arrows indicate the lengths and directions of the open reading frames, and the numbers in parentheses indicate their sizes. The PCR strategy is shown for BCG 1173P2 and M. tuberculosis 2296207. The small arrows refer to the positions of oligonucleotides C5 and C3. The first of the pair of numbers in boldface on the right refers to the total length of the amplified PCR product, whereas the second of the pair of numbers on the right refers to the size of the senX3-regX3 IR. The numbers on the bottom correspond to the sizes of the 3′ and 5′ ends of the senX3 and the regX3 genes, respectively. The senX3-regX3 IR of the two reference strains shown by the black arrows contain either two 77-bp MIRUs (larger arrows) or two 77-bp MIRUs and one 53-bp MIRU (smaller arrow). Only the AluI and BsrI restriction sites are shown.

FIG. 2

Analysis of the senX3-regX3 IR PCR products by agarose gel electrophoresis. DNA was isolated from M. microti (lane 2), M. bovis AN5 (lane 3), M. tuberculosis S200 (lane 4), M. africanum (lane 5), and M. bovis BCG (Glaxo) (lane 6) and was subjected to PCR amplification with oligonucleotides C5 and C3, and the PCR fragments were analyzed by electrophoresis with a 2.5% agarose gel, followed by staining with ethidium bromide. Lanes 7 and 8, PCR fragments with pRegX3Bc1 and pRegX3Mt1 as templates, respectively. The molecular size markers are in lanes 1 and 9, and the sizes of the PCR fragments are indicated in the left margin.

FIG. 3

Restriction analysis of the senX3-regX3 IR PCR products. The senX3-regX3 IRs from M. microti (lane 2), M. bovis AN5 (lane 3), M. tuberculosis S200 (lane 4), M. bovis BCG (Glaxo) (lane 5), pRegX3Mt1 (lane 6), and pRegX3Bc1 (lane 7) were amplified by PCR, digested with AluI and BsrI, subjected to polyacrylamide gel electrophoresis with an 8% polyacrylamide gel, and then stained with ethidium bromide. The molecular size markers are in lanes 1 and 8, and the sizes of the DNA fragments are indicated in the left margin.

FIG. 4

Distribution of the copy numbers of IS6110 among the M. tuberculosis strains tested. The copy numbers of IS6110 were estimated by RFLP analysis of the 109 clinical M. tuberculosis isolates that were subsequently tested by the senX3-regX3 IR PCR. Also included are the three M. tuberculosis strains which lack IS6110.

FIG. 5

Multiplex PCR analysis of three different mycobacterial strains. Chromosomal DNA was isolated from M. avium (lane 2), M. tuberculosis V.729 (lane 3), and M. tuberculosis S200 (lane 4) and subjected to multiplex PCR with primers C5 and C3, specific for the senX3-regX3 IR, and primers KY18 and KY98, specific for the 16S rRNA gene. The PCR products were then analyzed by electrophoresis with a 2.5% agarose gel and stained with ethidium bromide. Lane 5, PCR product amplified with pRegX3Mt1 as the template. The molecular size markers are in lanes 1 and 6, and the sizes of the PCR fragments are indicated in the left margin.