Evaluation of novel tandem repeat loci as potential Mycobacterium tuberculosis molecular typing VNTR regions

Abstract number: 1134_01_148

Thorne N., Gharbia S., Underwood A., Arnold C.

Objectives:  

The international community involved with molecular typing of M. tuberculosis isolates has recently focussed on Variable Number Tandem Repeat (VNTR) loci. VNTR-PCR involves amplification of the repeat region with primers in the flanking sequences. The number of repeat units is determined from the size of the product on an agarose gel. To date the majority of investigations into M. tuberculosis VNTRs have focussed on 40–100 bp repeat-unit VNTRs. In this study we looked at a range (3–78 bp repeats) including smaller repeat-unit VNTRs to investigate allelic variation in our collection of isolates and determine their usefulness in a molecular typing VNTR panel.

Methods:  

A collection of 23 isolates was used to investigate repeat-number variation at novel VNTR loci ranging from 3 bp to 78 bp in unit length. PCR of tandem repeat sequences were performed using primers in the 5’ and 3’ flanking regions. Pyrosequencing technology, non-denaturing High Performance Liquid Chromatography (non-dHPLC) and Beckman CEQ8000 fragment analysis were used to determine VNTR number for these loci. Beckman capillary sequencing was used to confirm VNTR copy number from sequence for anomalous fragment sizes and to further characterise the individual loci. VNTR copy number was determined from these fragment sizes using a simple calculation. These data were entered into Bionumerics software for phylogenetic analysis of isolates.

Results:  

Several novel VNTR loci tested were found to vary in copy number between strains in our isolate collection. The number of alleles varied for several of the novel VNTR loci. Certain loci were found to be useful to differentiate the ancient genotypic group one isolates from the more recent groups two and three. For example, all non-genogroup one isolates (n = 15) had one copy of VNTR12, while genogroup one isolates (n = 8) all had greater than one copy of this repeat. Further results characterising each novel locus will be presented.

Conclusion:  

Results from this study indicate that other tandem repeats, outside the 40–100 bp unit range, should be considered for investigations into highly discriminative markers for M. tuberculosis molecular typing. These investigations could help augment the final VNTR panel for an international strain-typing of M. tuberculosis. Our results may indicate that certain repeat loci within the genome could enable modelling of M. tuberculosis evolution and better inform the role of these repeat sequences in the chromosome.