Summary auto-generated
Frothingham and Meeker-O'Connell systematically analyzed variable number tandem repeat (VNTR) loci in the Mycobacterium tuberculosis complex as a novel strain differentiation method. They identified 11 tandem repeat loci in M. tuberculosis H37Rv: five major polymorphic tandem repeat (MPTR) loci containing 15-bp repeats and six exact tandem repeat (ETR) loci containing larger identical repeats. DNA from 48 strains, including 25 reference strains of M. tuberculosis, M. bovis, M. africanum, and M. microti, plus 23 M. bovis BCG substrains, was amplified at these loci using PCR. One MPTR locus and all six ETR loci showed length polymorphisms corresponding to insertions or deletions of repeat units. Combined analysis of the seven informative VNTR loci identified 22 distinct allele profiles among 25 wild-type strains and 5 profiles among 23 clonally derived BCG substrains. Allele profiles were reproducible and stable across multiple isolates obtained from different laboratories. The authors conclude that VNTR typing may be useful for bacterial strain differentiation and evolutionary studies, with potential applications including epidemiological investigations and outbreak identification.
Key findings
- Identification of 11 tandem repeat loci (5 MPTR and 6 ETR) in M. tuberculosis that demonstrate genetic variation through insertions and deletions of repeat units
- Seven VNTR loci generated 22 distinct allele profiles among 25 diverse wild-type tuberculosis complex strains, showing strong discriminatory power for strain differentiation
- VNTR typing proved more discriminative than IS6110 fingerprinting for low-copy-number strains such as M. bovis BCG substrains while being less discriminative for high-copy-number M. tuberculosis
- Reproducibility of VNTR typing demonstrated through multiple DNA samples of reference strains from different laboratories, all yielding identical results
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Abstract
Genetic loci containing variable numbers of tandem repeats (VNTR loci) form the basis for human gene mapping and identification, forensic analysis and paternity testing. The variability of bacterial tandem repeats has not been systematically studied. Eleven tandem repeat loci in the M. tuberculosis genome were analysed. Five major polymorphic tandem repeat (MPTR) loci contained 15-bp repeats with substantial sequence variation in adjacent copies. Six exact tandem repeat (ETR) loci contained large DNA repeats with identical sequences in adjacent repeats. These 11 loci were amplified in 48 strains to determine the number of tandem repeats at each locus. The strains analysed included 25 wild-type strains of M. tuberculosis, M. bovis, M. africanum and M. microti and 23 substrains of the attenuated M. bovis BCG vaccine. One of the five MPTR loci and all six ETR loci had length polymorphisms corresponding to insertions or deletions of tandem repeats. Most ETR loci were located in intergenic regions where copy number may influence expression of downstream genes. Each ETR locus had multiple alleles in the panel. Combined analysis identified 22 distinct allele profiles in 25 wild-type strains of the M. tuberculosis complex and five allele profiles in 23 M. bovis BCG substrains. Allele profiles were reproducible and stable, as demonstrated by analyses of multiple isolates of particular reference strains obtained from different laboratories. VNTR typing may be generally useful for strain differentiation and evolutionary studies in bacteria.