Skip to main content

Advertisement

SpringerLink
Log in

The Future of Digital Polymerase Chain Reaction in Virology

  • Review Article
  • Published:
Molecular Diagnosis & Therapy Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):263–73.

    Article  CAS  PubMed  Google Scholar 

  2. Bartlett JMS, Stirling D. A short history of the polymerase chain reaction. Methods Mol Biol. 2003;226:3–6.

    CAS  PubMed  Google Scholar 

  3. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y). 1992;10:413–7.

    Article  CAS  Google Scholar 

  4. Simmonds P, Balfe P, Peutherer JF, Ludlam CA, Bishop JO, Brown AJ. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol. 1990;64:864–72.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Morley AA. Digital PCR: a brief history. Biomol Detect Quantif. 2014;1:1–2.

    Article  Google Scholar 

  6. Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci. 1999;96:9236–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hayden RT, Gu Z, Sam SS, Sun Y, Tang L, Pounds S, et al. Comparative evaluation of three commercial quantitative cytomegalovirus standards by use of digital and real-time PCR. J Clin Microbiol. 2015;53:1500–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:45e.

    Article  Google Scholar 

  9. Trypsteen W, De Neve J, Bosman K, Nijhuis M, Thas O, Vandekerckhove L, et al. Robust regression methods for real-time polymerase chain reaction. Anal Biochem. 2015;480:34–6.

    Article  CAS  PubMed  Google Scholar 

  10. Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22.

    Article  CAS  PubMed  Google Scholar 

  11. Huggett JF, Foy CA, Benes V, et al. The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem. 2013;59:892–902.

    Article  CAS  PubMed  Google Scholar 

  12. Hayden RT, Gu Z, Ingersoll J, Abdul-Ali D, Shi L, Pounds S, et al. Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus. J Clin Microbiol. 2013;51:540–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Brunetto GS, Massoud R, Leibovitch EC, Caruso B, Johnson K, Ohayon J, et al. Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations. J Neurovirol. 2014;20:341–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Henrich TJ, Gallien S, Li JZ, Pereyra F, Kuritzkes DR. Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR. J Virol Methods. 2012;186(1–2):68–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kiselinova M, Pasternak AO, De Spiegelaere W, et al. Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA. PLoS One. 2014;9:e85999.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Strain MC, Lada SM, Luong T, Rought SE, Gianella S, Terry VH, et al. Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One. 2013;8:e55943.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. White RA, Quake SR, Curr K. Digital PCR provides absolute quantitation of viral load for an occult RNA virus. J Virol Methods. 2012;179:45–50.

    Article  CAS  PubMed  Google Scholar 

  18. Kiselinova M, Geretti AM, Malatinkova E, et al. HIV-1 RNA and HIV-1 DNA persistence during suppressive ART with PI-based or nevirapine-based regimens. J Antimicrob Chemother. 2015;70:3311–6.

    CAS  PubMed  Google Scholar 

  19. Kiselinova M, De Spiegelaere W, Buzon MJ, et al. Integrated and total HIV-1 DNA predict ex vivo viral outgrowth. PLoS Pathog. 2016;12:e1005472.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Mu D, Yan L, Tang H, Liao Y. A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system. Biotechnol Lett. 2015;37:2063–73.

    Article  CAS  PubMed  Google Scholar 

  21. Pavšič J, Žel J, Milavec M. Digital PCR for direct quantification of viruses without DNA extraction. Anal Bioanal Chem. 2016;408:67–75.

    Article  PubMed  Google Scholar 

  22. Kishida N, Noda N, Haramoto E, Kawaharasaki M, Akiba M, Sekiguchi Y. Quantitative detection of human enteric adenoviruses in river water by microfluidic digital polymerase chain reaction. Water Sci Technol. 2014;70:555.

    Article  CAS  PubMed  Google Scholar 

  23. Rački N, Morisset D, Gutierrez-Aguirre I, Ravnikar M. One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses. Anal Bioanal Chem. 2014;406:661–7.

    Article  PubMed  Google Scholar 

  24. Coudray-Meunier C, Fraisse A, Martin-Latil S, Guillier L, Delannoy S, Fach P, et al. A comparative study of digital RT-PCR and RT-qPCR for quantification of hepatitis A virus and norovirus in lettuce and water samples. Int J Food Microbiol. 2015;201:17–26.

    Article  CAS  PubMed  Google Scholar 

  25. Clementi M, Bagnarelli P. Are three generations of quantitative molecular methods sufficient in medical virology? Brief review. N Microbiol. 2015;38:437–41.

    Google Scholar 

  26. Hindson BJ, Ness KD, Masquelier DA, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011;83:8604–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pekin D, Skhiri Y, Baret J-C, et al. Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Lab Chip. 2011;11:2156.

    Article  CAS  PubMed  Google Scholar 

  28. Wang J, Ramakrishnan R, Tang Z, Fan W, Kluge A, Dowlati A, et al. Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays. Clin Chem. 2010;56:623–32.

    Article  CAS  PubMed  Google Scholar 

  29. Mukaide M, Sugiyama M, Korenaga M, Murata K, Kanto T, Masaki N, et al. High-throughput and sensitive next-generation droplet digital PCR assay for the quantitation of the hepatitis C virus mutation at core amino acid 70. J Virol Methods. 2014;207:169–77.

    Article  CAS  PubMed  Google Scholar 

  30. Taylor SC, Carbonneau J, Shelton DN, Boivin G. Optimization of droplet digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: clinical implications for quantification of oseltamivir-resistant subpopulations. J Virol Methods. 2015;224:58–66.

    Article  CAS  PubMed  Google Scholar 

  31. Whale AS, Bushell CA, Grant PR, et al. Detection of rare drug resistance mutations by digital PCR in a human influenza A virus model system and clinical samples. J Clin Microbiol. 2016;54:392–400.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sedlak RH, Cook L, Huang M-L, Magaret A, Zerr DM, Boeckh M, et al. Identification of Chromosomally integrated human herpesvirus 6 by droplet digital PCR. Clin Chem. 2014;60:765–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ota S, Ishikawa S, Takazawa Y, et al. Quantitative analysis of viral load per haploid genome revealed the different biological features of merkel cell polyomavirus infection in skin tumor. PLoS One. 2012;7:e39954.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. De Spiegelaere W, Erkens T, De Craene J, Burvenich C, Peelman L, Van den Broeck W. Elimination of amplification artifacts in real-time reverse transcription PCR using laser capture microdissected samples. Anal Biochem. 2008;382(1):72–4.

    Article  PubMed  Google Scholar 

  35. Suslov O, Steindler DA. PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency. Nucleic Acids Res. 2005;33:e181.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Sedlak RH, Kuypers J, Jerome KR. A multiplexed droplet digital PCR assay performs better than qPCR on inhibition prone samples. Diagn Microbiol Infect Dis. 2014;80:285–6.

    Article  CAS  PubMed  Google Scholar 

  37. Dingle TC, Sedlak RH, Cook L, Jerome KR. Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clin Chem. 2013;59:1670–2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Nixon G, Garson JA, Grant P, Nastouli E, Foy CA, Huggett JF. 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. Anal Chem. 2014;86:4387–94.

    Article  CAS  PubMed  Google Scholar 

  39. Malatinkova E, Kiselinova M, Bonczkowski P, Trypsteen W, Messiaen P, Vermeire J, et al. Accurate quantification of episomal HIV-1 two-long terminal repeat circles by use of optimized DNA isolation and droplet digital PCR. J Clin Microbiol. 2015;53:699–701.

    Article  CAS  PubMed  Google Scholar 

  40. Hoshino T, Inagaki F. Molecular quantification of environmental DNA using microfluidics and digital PCR. Syst Appl Microbiol. 2012;35:390–5.

    Article  CAS  PubMed  Google Scholar 

  41. Rački N, Dreo T, Gutierrez-Aguirre I, Blejec A, Ravnikar M. Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples. Plant Methods. 2014;10:42.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Yang R, Paparini A, Monis P, Ryan U. Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples. Int J Parasitol. 2014;44:1105–13.

    Article  CAS  PubMed  Google Scholar 

  43. Trypsteen W, Vynck M, De Neve J, Bonczkowski P, Kiselinova M, Malatinkova E, et al. ddpcRquant: threshold determination for single channel droplet digital PCR experiments. Anal Bioanal Chem. 2015;407:5827–34.

    Article  CAS  PubMed  Google Scholar 

  44. Hall Sedlak R, Jerome KR. The potential advantages of digital PCR for clinical virology diagnostics. Expert Rev Mol Diagn. 2014;14:501–07.

  45. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2012;84:1003–11.

    Article  CAS  PubMed  Google Scholar 

  46. Bosman KJ, Nijhuis M, van Ham PM, et al. Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir. Sci Rep. 2015;5:13811.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Sedlak RH, Cook L, Cheng A, Magaret A, Jerome KR. Clinical utility of droplet digital PCR for human cytomegalovirus. J Clin Microbiol. 2014;52:2844–8.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Coudray-Meunier C, Fraisse A, Martin-Latil S, et al. A novel high-throughput method for molecular detection of human pathogenic viruses using a nanofluidic real-time PCR system. PLoS One. 2016;11:e0147832.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Kiselinova M, De Spiegelaere W, Verhofstede C, Callens SFJ, Vandekerckhove L. Antiretrovirals for HIV prevention: when should they be recommended? Expert Rev Anti Infect Ther. 2014;12:431–45.

    Article  CAS  PubMed  Google Scholar 

  50. Sanders R, Mason DJ, Foy CA, et al. Evaluation of digital PCR for absolute RNA quantification. PLoS One. 2013;8:e75296.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Pavšič J, Devonshire AS, Parkes H, et al. Standardization of nucleic acid tests for clinical measurements of bacteria and viruses. J Clin Microbiol. 2015;53:2008–14.

    Article  PubMed  Google Scholar 

  52. Tang L, Sun Y, Buelow D, Gu Z, Caliendo AM, Pounds S, et al. Quantitative assessment of commutability for clinical viral load testing using a digital PCR-based reference standard. J Clin Microbiol. 2016;54:1616–23.

    Article  CAS  PubMed  Google Scholar 

  53. Haynes RJ, Kline MC, Toman B, Scott C, Wallace P, Butler JM, et al. Standard reference material 2366 for measurement of human cytomegalovirus DNA. J Mol Diagn. 2013;15:177–85.

    Article  CAS  PubMed  Google Scholar 

  54. Mattiuzzo G, Ashall J, Doris KS, et al. Development of lentivirus-based reference materials for ebola virus nucleic acid amplification technology-based assays. PLoS One. 2015;10:e0142751.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Dong L, Meng Y, Sui Z, Wang J, Wu L, Fu B. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material. Sci Rep. 2015;5:13174.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Jacobs BKM, Goetghebeur E, Clement L. Impact of variance components on reliability of absolute quantification using digital PCR. BMC Bioinform. 2014;15:283.

    Article  Google Scholar 

  57. Corbisier P, Pinheiro L, Mazoua S, Kortekaas A-M, Chung PYJ, Gerganova T, et al. DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials. Anal Bioanal Chem. 2015;407:1831–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Devonshire AS, Honeyborne I, Gutteridge A, Whale AS, Nixon G, Wilson P, et al. Highly reproducible absolute quantification of mycobacterium tuberculosis complex by digital PCR. Anal Chem. 2015;87:3706–13.

    Article  CAS  PubMed  Google Scholar 

  59. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods. 2013;10:1003–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Sedlak RH, Jerome KR. Viral diagnostics in the era of digital polymerase chain reaction. Diagn Microbiol Infect Dis. 2013;75:1–4.

    Article  CAS  PubMed  Google Scholar 

  61. Huggett JF, Cowen S, Foy CA. Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem. 2015;61:79–88.

    Article  CAS  PubMed  Google Scholar 

  62. Bhat S, Herrmann J, Armishaw P, Corbisier P, Emslie KR. Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Anal Bioanal Chem. 2009;394:457–67.

    Article  CAS  PubMed  Google Scholar 

  63. Huggett JF, Garson JA, Whale AS. Digital PCR and its potential application to microbiology. In: Persing DH, Tenover FC, Hayden RT, et al., editors. Molecular microbiology: diagnostic principles and practice, 3rd ed. Washington, DC; 2016. p. 49–57.

  64. Dorazio RM, Hunter ME. Statistical models for the analysis and design of digital polymerase chain reaction (dPCR) experiments. Anal Chem. 2015;87:10886–93.

    Article  CAS  PubMed  Google Scholar 

  65. Evans MI, Wright DA, Pergament E, Cuckle HS, Nicolaides KH. Digital PCR for noninvasive detection of aneuploidy: power analysis equations for feasibility. Fetal Diagn Ther. 2012;31:244–7.

    Article  PubMed  Google Scholar 

  66. Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S, et al. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res. 2012;40:e82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Kreutz JE, Munson T, Huynh T, Shen F, Du W, Ismagilov RF. Theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital PCR. Anal Chem. 2011;83:8158–68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Vynck M, Vandesompele J, Nijs N, Menten B, De Ganck A, Thas O. Flexible analysis of digital PCR experiments using generalized linear mixed models. Biomol Detect Quantif. 2016. doi:10.1016/j.bdq.2016.06.001.

  69. Lievens A, Jacchia S, Kagkli D, Savini C, Querci M. Measuring digital PCR quality: performance parameters and their optimization. PLoS One. 2016;11:e0153317.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Jones M, Williams J, Gärtner K, Phillips R, Hurst J, Frater J. Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, “definetherain”. J Virol Methods. 2014;202:46–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Dreo T, Pirc M, Ramšak Ž, Pavšič J, Milavec M, Žel J, et al. Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot. Anal Bioanal Chem. 2014;406:6513–28.

    Article  CAS  PubMed  Google Scholar 

  72. Dube S, Qin J, Ramakrishnan R. Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS One. 2008;3:e2876.

    Article  PubMed  PubMed Central  Google Scholar 

  73. Debski PR, Gewartowski K, Sulima M, Kaminski TS, Garstecki P. Rational design of digital assays. Anal Chem. 2015;87:8203–9.

    Article  CAS  PubMed  Google Scholar 

  74. Morisset D, Štebih D, Milavec M, Gruden K, Žel J. Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One. 2013;8:e62583.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Yu M, Carter KT, Makar KW, Vickers K, Ulrich CM, Schoen RE, et al. MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles. Epigenetics. 2015;10:803–9.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors thank the referees for their useful comments, which led to an improvement of the article.

Author contributions

Matthijs Vynck and Ward De Spiegelaere conceived the contents and structure of, and wrote, the manuscript. Wim Trypsteen, Olivier Thas, and Linos Vandekerckhove reviewed the manuscript. All authors agreed on the final version.

Author information

Authors and Affiliations

  1. Department of Mathematical Modelling, Statistics and Bioinformatics, Ghent University, Ghent, Belgium

    Matthijs Vynck & Olivier Thas

  2. HIV Translational Research Unit, Department of Internal Medicine, Ghent University, Depintelaan 185, 9000, Ghent, Belgium

    Wim Trypsteen, Linos Vandekerckhove & Ward De Spiegelaere

  3. National Institute for Applied Statistics Research Australia, School of Mathematics and Applied Statistics, University of Wollongong, Wollongong, NSW, Australia

    Olivier Thas

Authors
  1. Matthijs Vynck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wim Trypsteen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olivier Thas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Linos Vandekerckhove
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ward De Spiegelaere
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ward De Spiegelaere.

Ethics declarations

Conflict of interest

Matthijs Vynck, Wim Trypsteen, Olivier Thas, Linos Vandekerckhove, and Ward De Spiegelaere declare that they have no competing interests.

Funding

Ward De Spiegelaere is a post-doctoral fellow at the Research Foundation - Flanders (FWO), Grant Number 12G9716N; Linos Vanderkerckhove is a Senior Clinical Investigator at the FWO, Grant Number 1802014N; and Matthijs Vynck and Olivier Thas acknowledge the support of the Multidisciplinary Research Partnership Bioinformatics: From Nucleotides to Networks Project (01MR0310W) at Ghent University.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vynck, M., Trypsteen, W., Thas, O. et al. The Future of Digital Polymerase Chain Reaction in Virology. Mol Diagn Ther 20, 437–447 (2016). https://doi.org/10.1007/s40291-016-0224-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40291-016-0224-1

Keywords

Search

Navigation