Digital PCR

  • Birbal SinghEmail author
  • Gorakh Mal
  • Sanjeev K. Gautam
  • Manishi Mukesh


Precise detection of mutations is important for a variety of clinical and research applications. The digital PCR offers precise quantification of nucleic acids, viz. DNA, cDNA and RNA, and mutations. Digital PCR, used in the past also by different terms, is now at the apex of progression and applications in biological sciences owing to developments in chemical sciences, instrumentation, and accessibility of genome sequence data.
  • Highlights

  • Compared to qPCR, the digital PCR is more precise accurate for determining DNA and RNA quantities and mutations therein

  • Digital PCR is used in a variety of nucleic acid analytical and quantification methods.


Digital PCR Types of digital PCR Livestock applications Adulterations detection 


  1. Arendt M, Fall T, Lindblad-Toh K, Axelsson E (2014) Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes. Anim Genet 45(5):716–722. (Epub 2014 Jun 28)CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bosman KJ, Nijhuis M, van Ham PM, Wensing AM, Vervisch K, Vandekerckhove L, De Spiegelaere W (2015) Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir. Sci Rep. 9(5):13811. Scholar
  3. Brisco MJ, Condon J, Sykes PJ, Neoh SH, Morley AA (1991) Detection and quantitation of neoplastic cells in acute lymphoblastic leukaemia, by use of the polymerase chain reaction. Br J Haematol 79(2):211–217CrossRefGoogle Scholar
  4. Cawthorn D-M, Steinman HA, Hoffman LC (2013) A high incidence of species substitution and mislabelling detected in meat products sold in South Africa. Food Control 32:440–449CrossRefGoogle Scholar
  5. Chen R, Mias GI, Li-Pook-Than J, Jiang L, Lam HY, Chen R, Miriami E, Karczewski KJ, Hariharan M, Dewey FE, Cheng Y, Clark MJ, Im H, Habegger L, Balasubramanian S, O’Huallachain M, Dudley JT, Hillenmeyer S, Haraksingh R, Sharon D, Euskirchen G, Lacroute P, Bettinger K, Boyle AP, Kasowski M, Grubert F, Seki S, Garcia M, Whirl-Carrillo M, Gallardo M, Blasco MA, Greenberg PL, Snyder P, Klein TE, Altman RB, Butte AJ, Ashley EA, Gerstein M, Nadeau KC, Tang H, Snyder M (2012) Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell 148(6):1293–1307. Scholar
  6. Conte D, Verri C, Borzi C, Suatoni P, Pastorino U, Sozzi G, Fortunato O (2015) Novel method to detect microRNAs using chip-based QuantStudio 3D digital PCR. BMC Genom 23(16):849. Scholar
  7. Cremonesi P, Cortimiglia C, Picozzi C, Minozzi G, Malvisi M, Luini M, Castiglioni B (2016) Development of a droplet digital polymerase chain reaction for rapid and simultaneous identification of common foodborne pathogens in soft cheese. Front Microbiol 7:1725 (eCollection 2016)Google Scholar
  8. D’Aversa E, Breveglieri G, Pellegatti P, Guerra G, Gambari R, Borgatti M (2018) Non-invasive fetal sex diagnosis in plasma of early weeks pregnants using droplet digital PCR. Mol Med 24(1):14. Scholar
  9. Dingle TC, Sedlak RH, Cook L, Jerome KR (2013) Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clin Chem 59(11):1670–1672. (Epub 2013 Sep 3)CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dong G, Meng F, Zhang Y, Cui Z, Lidan H, Chang S, Zhao P (2018) Development and evaluation of a droplet digital PCR assay for the detection of fowl adenovirus serotypes 4 and 10 in attenuated vaccines. J Virol Methods. pii: S0166-0934(18)30417-8. (Epub ahead of print)CrossRefGoogle Scholar
  11. Elmahalawy ST, Halvarsson P, Skarin M, Höglund J (2018) Droplet digital polymerase chain reaction (ddPCR) as a novel method for absolute quantification of major gastrointestinal nematodes in sheep. Vet Parasitol 15(261):1–8. (Epub 2018 Jul 18)CrossRefGoogle Scholar
  12. Flatschart RB, Almeida DO, Heinemann MB, Medeiros MN, Granjeiro JM, Folgueras-Flatschart AV (2015) Absolute quantification of Bovine Viral Diarrhea Virus (BVDV) RNA by the digital PCR technique. J Phys Conf Ser 575(1):012038 (IOP Publishing)Google Scholar
  13. Frésard L, Leroux S, Servin B, Gourichon D, Dehais P, Cristobal MS, Marsaud N, Vignoles F, Bed’hom B, Coville JL, Hormozdiari F, Beaumont C, Zerjal T, Vignal A, Morisson M, Lagarrigue S, Pitel F (2014) Transcriptome-wide investigation of genomic imprinting in chicken. Nucleic Acids Res 42(6):3768–3782. (Epub 2014 Jan 21)CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gerdes L, Iwobi A, Busch U, Pecoraro S (2016) Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms. Biomol Detect Quantif 7:9–20. (eCollection 2016 Mar)CrossRefGoogle Scholar
  15. Gou T, Hu J, Wu W, Ding X, Zhou S, Fang W, Mu Y (2018) Smartphone-based mobile digital PCR device for DNA quantitative analysis with high accuracy. Biosens Bioelectron 30(120):144–152. (Epub 2018 Aug 17)CrossRefGoogle Scholar
  16. Guo G, Huss M, Tong GQ, Wang C, Li Sun L, Clarke ND, Robson P (2010) Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev Cell 18(4):675–685. Scholar
  17. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83(22):8604–8610. (Epub 2011 Oct 28)CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL, Tewari M (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10(10):1003–1005. (Epub 2013 Sep 1)CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kuypers J, Jerome KR (2017) Applications of digital PCR for clinical microbiology. J Clin Microbiol 55(6):1621–1628. (Epub 2017 Mar 15. Review)CrossRefGoogle Scholar
  20. Lai YC, Fujikawa T, Maemura T, Ando T, Kitahara G, Endo Y, Yamato O, Koiwa M, Kubota C, Miura N (2017) Inflammation-related microRNA expression level in the bovine milk is affected by mastitis. PLoS ONE 12(5):e0177182. (eCollection 2017)CrossRefPubMedPubMedCentralGoogle Scholar
  21. Lo YM, Lun FM, Chan KC, Tsui NB, Chong KC, Lau TK, Leung TY, Zee BC, Cantor CR, Chiu RW (2007) Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci U S A. 104(32):13116–13121CrossRefGoogle Scholar
  22. Ludlow AT, Robin JD, Sayed M, Litterst CM, Shelton DN, Shay JW, Wright WE (2014) Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution. Nucleic Acids Res 42(13):e104. Scholar
  23. Ludlow AT, Shelton D, Wright WE, Shay JW (2018) ddTRAP: a method for sensitive and precise quantification of telomerase activity. Methods Mol Biol 1768:513–529. Scholar
  24. Miotto E, Saccenti E, Lupini L, Callegari E, Negrini M, Ferracin M (2014) Quantification of circulating miRNAs by droplet digital PCR: comparison of EvaGreen- and TaqMan-based chemistries. Cancer Epidemiol Biomarkers Prev 23(12):2638–2642. Scholar
  25. Morley AA (2014) Digital PCR: a brief history. Biomol Detect Quantif 1(1):1–2 (eCollection 2014 Sep)CrossRefGoogle Scholar
  26. Morisset D, Štebih D, Milavec M, Gruden K, Žel J (2013) Quantitative analysis of food and feed samples with droplet digital PCR. PLoS ONE 8(5):e62583. (Print 2013)CrossRefPubMedPubMedCentralGoogle Scholar
  27. O’Mahony PJ (2013) Finding horse meat in beef products—a global problem. QJM 106(6):595–597. Scholar
  28. Paquette SJ, Stanford K, Thomas J, Reuter T (2018) Quantitative surveillance of shiga toxins 1 and 2, Escherichia coli O178 and O157 in feces of western-Canadian slaughter cattle enumerated by droplet digital PCR with a focus on seasonality and slaughterhouse location. PLoS One 13(4):e0195880. (eCollection 2018)CrossRefGoogle Scholar
  29. Pendleton AL, Shen F, Taravella AM, Emery S, Veeramah KR, Boyko AR, Kidd JM (2018) Comparison of village dog and wolf genomes highlights the role of the neural crest in dog domestication. BMC Biol 16(1):64. Scholar
  30. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emslie KR (2012) Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 84(2):1003–1011. (Epub 2011 Dec 21)CrossRefPubMedGoogle Scholar
  31. Quigley L, O’Sullivan O, Stanton C, Beresford TP, Ross RP, Fitzgerald GF, Cotter PD (2013) The complex microbiota of raw milk. FEMS Microbiol Rev 37(5):664–698. Scholar
  32. Ren J, Deng T, Huang W, Chen Y, Ge Y (2017) A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food. PLoS One 12(3):e0173567. (eCollection 2017)CrossRefGoogle Scholar
  33. Robinson S, Follo M, Haenel D, Mauler M, Stallmann D, Heger LA, Helbing T, Duerschmied D, Peter K, Bode C, Ahrens I, Hortmann M (2018) Chip-based digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction patients. Acta Pharmacol Sin 39(7):1217–1227. Scholar
  34. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491CrossRefGoogle Scholar
  35. Simmonds P, Balfe P, Peutherer JF, Ludlam CA, Bishop JO, Brown AJ (1990) Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol 64(2):864–872PubMedPubMedCentralGoogle Scholar
  36. Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA (1992) Quantitation of targets for PCR by use of limiting dilution. Biotechniques 13(3):444–449PubMedGoogle Scholar
  37. Tadmor AD, Ottesen EA, Leadbetter JR, Phillips R (2011) Probing individual environmental bacteria for viruses by using microfluidic digital PCR. Science 333(6038):58–62. Scholar
  38. Tang SP, Zhang Y, Huang JH (2016) Analysis of beef and lamp products adulteration in Guangdong province. J Food Saf Qual 7(5):1882–1886 (ref. 15)Google Scholar
  39. Tian Q, Mu Y, Xu Y, Song Q, Yu B, Ma C, Jin W, Jin Q (2015) An integrated microfluidic system for bovine DNA purification and digital PCR detection. Anal Biochem 15(491):55–57. Scholar
  40. Verhaegen B, De Reu K, De Zutter L, Verstraete K, Heyndrickx M, Van Coillie E (2016) Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces. Toxins (Basel) 8(5). pii: E157. Scholar
  41. Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A. 96(16):9236–9241CrossRefGoogle Scholar
  42. Wang J, Ramakrishnan R, Tang Z, Fan W, Kluge A, Dowlati A, Jones RC, Ma PC (2010) Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays. Clin Chem 56(4):623–632. Scholar
  43. Wang Q, Zhang B, Xu X, Long F, Wang J (2018) CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method. Sci Rep. 8(1):14126. Scholar
  44. Yung TK, Chan KC, Mok TS, Tong J, To KF, Lo YM (2009) Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients. Clin Cancer Res 15(6):2076–2084. Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Birbal Singh
    • 1
    Email author
  • Gorakh Mal
    • 1
  • Sanjeev K. Gautam
    • 2
  • Manishi Mukesh
    • 3
  1. 1.ICAR-Indian Veterinary Research Institute, Regional StationPalampurIndia
  2. 2.Department of BiotechnologyKurukshetra UniversityKurukshetraIndia
  3. 3.Department of Animal BiotechnologyICAR-National Bureau of Animal Genetic ResourcesKarnalIndia

Personalised recommendations