Highly reproducible absolute quantification of Mycobacterium tuberculosis 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 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.

[1]  Alison S. Devonshire,et al.  Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification , 2014, Analytical and Bioanalytical Chemistry.

[2]  Alexandra S. Whale,et al.  Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples , 2013, PloS one.

[3]  E. Houpt,et al.  Digital PCR to Detect and Quantify Heteroresistance in Drug Resistant Mycobacterium tuberculosis , 2013, PloS one.

[4]  Ramesh Ramakrishnan,et al.  Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution. , 2010, Methods.

[5]  P. V. van Helden,et al.  Molecular Bacterial Load Assay, a Culture-Free Biomarker for Rapid and Accurate Quantification of Sputum Mycobacterium tuberculosis Bacillary Load during Treatment , 2011, Journal of Clinical Microbiology.

[6]  Jim F Huggett,et al.  Evaluation of digital PCR for absolute DNA quantification. , 2011, Analytical chemistry.

[7]  Stephen R Quake,et al.  Digital PCR provides sensitive and absolute calibration for high throughput sequencing , 2008, BMC Genomics.

[8]  Jim F Huggett,et al.  Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus. , 2014, Analytical chemistry.

[9]  Stephen R Quake,et al.  Digital PCR provides absolute quantitation of viral load for an occult RNA virus. , 2012, Journal of virological methods.

[10]  Alexandra S. Whale,et al.  Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation , 2012, Nucleic acids research.

[11]  Kerry R Emslie,et al.  Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units. , 2010, Analytical chemistry.

[12]  V. Cheng,et al.  Molecular diagnostics in tuberculosis , 2005, European Journal of Clinical Microbiology and Infectious Diseases.

[13]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.

[14]  Tania Nolan,et al.  The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. , 2013, Clinical chemistry.

[15]  Martin S. Taylor,et al.  Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis. , 2013, Methods.

[16]  Blaza Toman,et al.  Standard reference material 2366 for measurement of human cytomegalovirus DNA. , 2013, The Journal of molecular diagnostics : JMD.

[17]  Phillip Belgrader,et al.  Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay , 2013, Journal of Clinical Microbiology.

[18]  T. Dingle,et al.  Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. , 2013, Clinical chemistry.

[19]  Philippe Corbisier,et al.  Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number , 2009, Analytical and bioanalytical chemistry.

[20]  J. Wong,et al.  Advantages of using the QIAshredder instead of restriction digestion to prepare DNA for droplet digital PCR. , 2014, BioTechniques.

[21]  J. Rodríguez,et al.  Comparison of methods of DNA extraction for real‐time PCR in a model of pleural tuberculosis , 2010, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[22]  G. Woods,et al.  Comparison of Six Methods of Extracting Mycobacterium tuberculosis DNA from Processed Sputum for Testing by Quantitative Real-Time PCR , 2005, Journal of Clinical Microbiology.

[23]  Benjamin J. Hindson,et al.  Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification , 2011, Analytical chemistry.

[24]  Z. Gu,et al.  Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus , 2012, Journal of Clinical Microbiology.

[25]  Douglas D. Richman,et al.  Highly Precise Measurement of HIV DNA by Droplet Digital PCR , 2013, PloS one.

[26]  Lianhua Dong,et al.  Erratum to: Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers , 2014, Analytical and Bioanalytical Chemistry.

[27]  P. Corbisier,et al.  Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction , 2010, Analytical and bioanalytical chemistry.

[28]  Hai Wu,et al.  Rapid detection and monitoring therapeutic efficacy of Mycobacterium tuberculosis complex using a novel real-time assay. , 2012, Journal of microbiology and biotechnology.

[29]  Sebastien Gallien,et al.  Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR. , 2012, Journal of virological methods.

[30]  Kerry R Emslie,et al.  Effect of sustained elevated temperature prior to amplification on template copy number estimation using digital polymerase chain reaction. , 2011, The Analyst.

[31]  Elizabeth McCarthy,et al.  Development and Evaluation of a Next-Generation Digital PCR Diagnostic Assay for Ocular Chlamydia trachomatis Infections , 2013, Journal of Clinical Microbiology.

[32]  Maja Ravnikar,et al.  One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses , 2013, Analytical and Bioanalytical Chemistry.