Optimization of standard in-house 24-locus variable-number tandem-repeat typing for Mycobacterium tuberculosis and its direct application to clinical material
| Autor: | Dick van Soolingen, Adri G. M. van der Zanden, Jakko van Ingen, Anita C. Schürch, Arnout Mulder, Onno W. Akkerman, Jessica L. de Beer, Tjip S. van der Werf |
|---|---|
| Přispěvatelé: | Microbes in Health and Disease (MHD) |
| Rok vydání: | 2014 |
| Předmět: |
Microbiology (medical)
Genetics COMPLEX Serial dilution Genotype Mycobacterium tuberculosis Multiple Loci VNTR Analysis Biology bacterial infections and mycoses biology.organism_classification Polymerase Chain Reaction law.invention Molecular Typing Variable number tandem repeat lnfectious Diseases and Global Health Radboud Institute for Health Sciences [Radboudumc 4] Mycobacterium tuberculosis complex law Genetic Loci Tandem Repeat Sequences Multilocus sequence typing Typing Letters to the Editor Polymerase chain reaction DNA Primers |
| Zdroj: | Journal of Clinical Microbiology, 52, 1338-42 Journal of Clinical Microbiology, 52, 5, pp. 1338-42 Journal of Clinical Microbiology, 52(5), 1338-1342. AMER SOC MICROBIOLOGY |
| ISSN: | 0095-1137 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|