title: Optimization of standard in-house 24-locus variable-number tandem-repeat typing for Mycobacterium tuberculosis and its direct application to clinical material.
abstract: 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.
authors: National Tuberculosis Reference Laboratory (IDS), Centre for Infectious Disease Control (CIB), National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
authors: Jessica L
authors: JL
authors: de Beer
authors: Onno W
authors: OW
authors: Akkerman
authors: Anita C
authors: AC
authors: Schürch
authors: Arnout
authors: A
authors: Mulder
authors: Tjip S
authors: TS
authors: van der Werf
authors: Adri G M
authors: AG
authors: van der Zanden
authors: Jakko
authors: J
authors: van Ingen
authors: Dick
authors: D
authors: van Soolingen
mesh_terms: D017931:DNA Primers / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics*; D005838:Genotype; D058889:Molecular Typing / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020080:Tandem Repeat Sequences / Q000235:genetics*
chemical_list: D017931:DNA Primers