title: Highly reproducible absolute quantification of Mycobacterium tuberculosis complex by digital PCR.
abstract: 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 Mycobacterium tuberculosis 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.
authors: †Molecular and Cell Biology Team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.
authors: Alison S
authors: AS
authors: Devonshire
authors: ‡Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, University College London, London NW3 2PF, United Kingdom.
authors: Isobella
authors: I
authors: Honeyborne
authors: †Molecular and Cell Biology Team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.
authors: Alice
authors: A
authors: Gutteridge
authors: †Molecular and Cell Biology Team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.
authors: Alexandra S
authors: AS
authors: Whale
authors: †Molecular and Cell Biology Team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.
authors: Gavin
authors: G
authors: Nixon
authors: §Statistics Team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.
authors: Philip
authors: P
authors: Wilson
authors: †Molecular and Cell Biology Team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.
authors: Gerwyn
authors: G
authors: Jones
authors: ‡Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, University College London, London NW3 2PF, United Kingdom.
authors: Timothy D
authors: TD
authors: McHugh
authors: †Molecular and Cell Biology Team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.
authors: Carole A
authors: CA
authors: Foy
authors: †Molecular and Cell Biology Team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.|‡Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, University College London, London NW3 2PF, United Kingdom.
authors: Jim F
authors: JF
authors: Huggett
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D006801:Humans; D009164:Mycobacterium Infections / Q000382:microbiology*; D009163:Mycobacterium bovis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D004269:DNA, Bacterial