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
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