Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis

Abstract

Although the virulences and host ranges differ among members of the Mycobacterium tuberculosis complex (TBC; M. tuberculosis, M. africanum, M. canettii, M. microti, M. bovis, and M. bovis BCG), commercially available molecular assays cannot differentiate these organisms because of the genetic identities of their 16S rRNA gene sequences. Comparative genomic analyses with the complete DNA sequence of M. tuberculosis H37Rv has provided information on regions of difference (RD 1 to RD 16) deleted in members of the TBC other than M. tuberculosis. To determine whether deletion analysis could accurately differentiate members of TBC, we used PCR to assess the presence or absence of specific regions of the genome in 88 well-characterized isolates of M. tuberculosis, M. africanum, M. microti, M. bovis, and M. bovis BCG. The identifications obtained by use of the specific deletion profiles correlated 100% with the original identifications for all TBC members except M. africanum, but further characterization resulted in profiles specific for all members. Although six RD regions were used in the analyses with the original 88 isolates, it was found that the use of RD 1, RD 9, and RD 10 was sufficient for initial screenings, followed by the use of RD 3, RD 5, and RD 11 if the results for any of the first three regions were negative. When 605 sequential clinical isolates were screened, 578 (96%) were identified as M. tuberculosis, 6 (1%) were identified as M. africanum, 8 (1%) were identified as M. bovis, and 13 (2%) were identified as M. bovis BCG. Since PCR-based assays can be implemented in most clinical mycobacteriology laboratories, this approach provides a rapid and simple means for the differentiation of members of TBC, especially M. bovis and M. tuberculosis, when it is important to distinguish between zoonotic sources (i.e., cattle and unpasteurized dairy products) and human sources of tuberculosis disease.

FIG. 1.

Results of PCR for presence of RD 9 and RD 10. The larger PCR products indicate the presence of RD 9 (upper lanes) or RD 10 (lower lanes), while the smaller products indicate that the region has been deleted. The size standards (in base pairs) are labeled on the left side of the same 2% agarose gel prepared with a double comb.

FIG. 2.

Oxygen preference test (Middlebrook 7H9 broth with 0.2% agar). Four different members of the M. tuberculosis complex are shown, with the oxygen preference typical for each member indicated. Tube 1, M. tuberculosis; tube 2, M. africanum; tube 3, M. bovis; tube 4, M. bovis BCG.

FIG. 3.

Flow chart for identification of TBC isolates by deletion analysis.