Abstract
BackgroundMolecular genotyping of M.tuberculosis is an important laboratory tool in the context of emerging drug resistant TB. The standard 24-loci MIRU-VNTR typing includes PCR amplification followed by the detection and sizing of PCR fragments using capillary electrophoresis on automated sequencers or using agarose gels. The QIAxcel Advanced system might offer a cost-effective medium-throughput alternative.MethodsPerformance characteristics of the QIAxcel Advanced platform for the standard 24 VNTR loci panel was evaluated at two centres on a total of 140 DNA specimens using automated capillary electrophoresis as a reference method. Additionally 4 hypervariable MIRU-VNTR loci were evaluated on 53 crude DNA extracts. The sizing accuracy, interlaboratory reproducibility and overall instrument’s performance were assessed during the study.ResultsAn overall concordance with the reference method was high reaching 98.5% and 97.6% for diluted genomic and crude DNA extracts respectively. 91.4% of all discrepancies were observed in fragments longer than 700bp. The concordance for hypervariable loci was lower except for locus 4120 (96.2%). The interlaboratory reproducibility agreement rates were 98.9% and 91.3% for standard and hypervariable loci, respectively. Overall performance of the QIAxcel platform for M.tuberculosis genotyping using a panel of standard loci is comparable to that of established methods for PCR fragments up to 700bp. Inaccuracies in sizing of longer fragments could be resolved through using in-house size markers or introduction of offset values. To conclude, the QiaXcel system could be considered an effective alternative to existing methods in smaller reference and regional laboratories offering good performance and shorter turnaround times.
Highlights
Molecular genotyping of M.tuberculosis is an important laboratory tool in the context of emerging drug resistant TB
All panels included M. tuberculosis strains from various lineages; panels 2 and 3 were enriched with Beijing family strains to test the performance of the system for highly conserved genotyped using hypervariable loci
Performance of the assay for a standard 24 VNTR loci was assessed at both centres using two panels of M. tuberculosis DNA specimens
Summary
Molecular genotyping of M.tuberculosis is an important laboratory tool in the context of emerging drug resistant TB. The standard 24-loci MIRU-VNTR typing includes PCR amplification followed by the detection and sizing of PCR fragments using capillary electrophoresis on automated sequencers or using agarose gels. The QIAxcel Advanced system might offer a cost-effective medium-throughput alternative.
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