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

Devonshire, Alison S.; Honeyborne, Isobella; Gutteridge, Alice; Whale, Alexandra S.; Nixon, Gavin; Wilson, Philip J.; Jones, Gerwyn M.; McHugh, Timothy D.; Foy, Carole A.; Huggett, Jim F.

doi:10.1021/ac5041617

Cited by 90 publications

(88 citation statements)

References 35 publications

(73 reference statements)

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“…In addition, assay design (e.g. primer pairs), master mix choice, extraction method, etc., may cause bias [58]. Apart from an improvement of current dPCR platforms, improvements in design and analysis methods will help us better cope with these issues.…”

Section: Quantitative Accuracymentioning

confidence: 99%

The Future of Digital Polymerase Chain Reaction in Virology

et al. 2016

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Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a tool for the quantification of nucleic acids. The current technologies are more precise and accurate, but may not be much more sensitive, compared with quantitative PCR (qPCR) applications. The most promising applications with the current technology are the analysis of mutated sequences, such as emerging drug-resistant mutations. Guided by the recent literature, this review focuses on three aspects that demonstrate the potential of dPCR for virology researchers and clinicians: the applications of dPCR within both virology research and clinical virology, the benefits of the technique over the currently used real-time qPCR, and the importance and availability of specific data analysis approaches for dPCR. Comments are provided on current drawbacks and often overlooked pitfalls that need further attention to allow widespread implementation of dPCR as an accurate and precise tool within the field of virology.

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Section: Quantitative Accuracymentioning

confidence: 99%

The Future of Digital Polymerase Chain Reaction in Virology

et al. 2016

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“…The discrete nature of compartments enables digital quantification of absolute numbers of nucleic acids in a sample,3 accurate estimation of copy‐number variation,4 detection of pathogens5 and rare cancer mutations,6 as well as other applications 7…”

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confidence: 99%

DNA Nanoparticles for Improved Protein Synthesis In Vitro

et al. 2016

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The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

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“…Although the theory of dPCR assumes that each target nucleic acid molecule present in a partition of the BioMark dPCR chip (Fluidigm) will undergo successful amplification, this study and many others have shown that more careful method design and optimisation are required to obtain an accurate quantitative result [6], [7], [23], [24]. The uncertainties observed demonstrate that precise and reproducible quantification of extracted genomic- as well as synthetic RNA is achievable by RT-dPCR.…”

Section: Discussionmentioning

confidence: 82%

“…It has been successfully applied in the certification of reference materials used for standardising qPCR assays widely employed in clinical diagnostics and research areas [2], [3], [4]. Despite these major advantages, several studies have also reported significant bias when measuring both DNA and/or RNA using dPCR [5], [6], [7]. Such discrepancies have been attributed to the choice of the dPCR format (chamber vs droplet-based dPCR), the complexity of the nucleic acid template, detection reagents used, and pre-analytical steps such as nucleic acid extraction [3], [4].…”

Section: Introductionmentioning

confidence: 99%

Influence of primer & probe chemistry and amplification target on reverse transcription digital PCR quantification of viral RNA

Heuverswyn

Karczmarczyk

Schimmel

et al. 2016

Biomolecular Detection and Quantification

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Compared to other PCR technologies, digital PCR is a potentially highly accurate approach for the quantification of nucleic acid fragments. This study describes the impact of four experimental factors, namely primer and probe chemistry, PCR amplification target, duplexing, and template type, on the measurement results obtained by reverse transcription digital PCR (RT-dPCR) of viral RNA using influenza A virus as a model. Along conventional dual labelled probes (DLP), alternative primer and probe chemistries, including Zip Nucleic Acids (ZNAs), Locked Nucleic Acids (LNAs), and Scorpions®, were compared with two RNA template types: i) total genomic RNA extracted from cell cultured influenza A and ii) a synthetically prepared RNA transcript (In vitro transcribed RNA).While apparently duplexing or a different PCR target choice did not have a significant influence on the estimated RNA copy numbers, the impact of the choice of primer and probe chemistry and template type differed significantly for some methods. The combined standard uncertainty of the dPCR analysis results has been assessed, taking into account both the repeatability and the intermediate precision of the procedure.Our data highlight the importance of dPCR method optimisation and the advantage of using a more sophisticated primer and probe chemistry, which turned out to be dependent on the template type. Considerations are provided with respect to the molecular diagnostics of viral RNA pathogens, and more specifically, for precise quantification of RNA, which is of tremendous importance for the development of RNA calibration materials and the qualification of these calibrants as certified reference materials.

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Highly Reproducible Absolute Quantification of Mycobacterium tuberculosis Complex by Digital PCR

Cited by 90 publications

References 35 publications

The Future of Digital Polymerase Chain Reaction in Virology

The Future of Digital Polymerase Chain Reaction in Virology

DNA Nanoparticles for Improved Protein Synthesis In Vitro

Influence of primer & probe chemistry and amplification target on reverse transcription digital PCR quantification of viral RNA

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