Optimization of standard in-house 24-locus variable-number tandem-repeat typing for Mycobacterium tuberculosis and its direct application to clinical material
Journal of Clinical Microbiology , Volume 52 - Issue 5 p. 1338- 1342
Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable number of single-locus PCRs have to be performed for the loci with missing results, which impairs the laboratory work flow. Therefore, the original in-house method described by Supply et al. in 2006 was reevaluated. We modified seven primers and the PCR master mixture and obtained a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns detected in the routine flow of typing activities increased to 84.7% from the 72.3% obtained with the typing conducted with the commercially available Genoscreen MIRU-VNTR typing kit. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of M. tuberculosis in bronchoalveolar lavage fluid. A 1:10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern. The optimized in-house 24-locus VNTR typing method will reduce the turnaround time of typing significantly and also the financial burden of these activities.
|Journal of Clinical Microbiology|
|Organisation||Department of Virology|
de Beer, J.L, Akkerman, O.W, Schürch, A, Mulder, A, van der Werf, T.S, van der Zanden, A, … van Soolingen, D. (2014). Optimization of standard in-house 24-locus variable-number tandem-repeat typing for Mycobacterium tuberculosis and its direct application to clinical material. Journal of Clinical Microbiology, 52(5), 1338–1342. doi:10.1128/JCM.03436-13