Highly Reproducible Absolute Quantification of <i>Mycobacterium tuberculosis</i> Complex by Digital PCR

Digital PCR (dPCR) offers absolute quantification through the limiting dilution of template nucleic acid molecules and has the potential to offer high reproducibility. However, the robustness of dPCR has yet to be evaluated using complex genomes to compare different dPCR methods and platforms. We used DNA templates from the pathogen <i>Mycobacterium tuberculosis</i> to evaluate the impact of template type, master mixes, primer pairs and, crucially, extraction methods on dPCR performance. Performance was compared between the chip (BioMark) and droplet (QX100) formats. In the absence of any external calibration, dPCR measurements were generally consistent within ∼2-fold between different master mixes and primers. Template DNA integrity could influence dPCR performance: high molecular weight gDNA resulted in underperformance of one master mix, while restriction digestion of a low molecular weight sample also caused underestimation. Good concordance (≤1.5-fold difference) was observed between chip and droplet formats. Platform precision was in agreement with predicted Poisson error based on partition number, but this was a minor component (<10%) of the total variance when extraction was included. dPCR offers a robust reproducible method for DNA measurement; however, as a predominant source of error, the process of DNA extraction will need to be controlled with suitable calibrators to maximize agreement between laboratories

Additional file 5: of Profiling persistent tubercule bacilli from patient sputa during therapy predicts early drug efficacy

Figure S1. An illustration of the computation process to test associations between transcriptional signatures and patient variables. Figure S2. Venn diagrams describing the overlapping transcriptional signatures of bacilli in sputum relative to aerobic log phase growth. Figure S3. Box and whisker plots mapping the differential expression of gene families. Figure S4. Hierarchical clustering of the mean M.tb transcriptional profiles derived from sputa. Figure S5. Venn diagram highlighting genes significantly induced 3 days after the start of drug therapy. Figure S6. Contrasting patient trajectories as defined by principle component analysis plotting day 0 and day 3 timepoints only. (PDF 526 kb

Additional file 2: of The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

Protocol for gDNA extraction and dPCR quantification of materials. (PDF 394 kb