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Progress in Chemistry 2020, Vol. 32 Issue (5): 581-593 DOI: 10.7536/PC190913 Previous Articles   Next Articles

• Review •

Development and Application of Digital PCR Technology

Huitiao Li1, Jianzhang Pan1,**(), Qun Fang1,**()   

  1. Institute of Microanalytical Systems, Department of Chemistry and Center for Chemistry of Novel & High-Performance Materials, Zhejiang University, Hangzhou 310058, China
  • Received: Revised: Online: Published:
  • Contact: Jianzhang Pan, Qun Fang
  • About author:
    ** e-mail: (Qun Fang);
    (Jianzhang Pan)
  • Supported by:
    National Natural Science Foundation of China(21974122); National Natural Science Foundation of China(21827806); National Natural Science Foundation of China(21435004)
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Digital PCR(dPCR) assay is a highly sensitive absolute quantitative analysis technique for nucleic acids. Digital PCR systems perform amplification by equally dividing the reaction mixtures into a large number of independent reaction units, and calculates the nucleic acid copy number based on the Poisson distribution and the positive ratio. With the advantages of high sensitivity, accuracy and tolerance, digital PCR assay enables absolute quantitative analysis of samples. In recent years, with the maturity of microfluidic technology, digital PCR assay based on microfluidic technology has been rapidly developed, and widely applied in the analysis of gene mutation, copy number variation, viral microbial, genetically modified foods and DNA sequencing. In this article, the principle, development and applications of the digital PCR technique are reviewed.

Contents

1 Introduction

2 Principle of dPCR technology

3 Development and classification of dPCR technology

3.1 Microchamber-based digital PCR assay

3.2 Microfluidic chip-based digital PCR assay

3.3 Droplet-based digital PCR assay

4 Applications of dPCR technology

4.1 Single cell genetic analysis

4.2 Tumour research

4.3 Prenatal diagnosis

4.4 Virus and microbial analysis

4.5 Food safety and environmental monitoring

4.6 Next generation sequencing validation and gene editing

5 Conclusion and outlook

Key words: microfluidics technology, digital PCR, absolute quantification
Fig. 1 The principle of Digital PCR assay[8]
Fig. 2 Numbers of published articles in the field of digital PCR from 1999 to 2018 in Web of Science
Fig. 3 Microhole array-based digital PCR chip (a) stainless steel digital PCR chip[14];(b) QuantstudioTM 3D digital PCR chip[15];(c) ClarityTM digital PCR chip[16];(d) Constellation digital PCR chip[17]
Fig. 4 (a) Digital PCR chip with PDMS microvalve structure and nucleic acid amplification results[19];(b)digital PCR chip with femtoliter microreaction chamber[20]
Fig. 5 (a)Digital PCR chip with millions of picoliters microreaction chambers[21];(b)self-digitization digital PCR chip[23];(c)self-priming compartmentalization digital PCR chip[24];(d)PET spinning disk digital PCR chip[25]
Fig. 6 Schematic diagram of BEAMing technique[28]
Fig. 7 Digital PCR analysis system integrated droplet formation, droplet reinjection, electron droplet fusion, and online fluorescence detection[33, 34]
Fig. 8 Digital PCR system integrating process of droplet formation, splitting, arraying, thermal cycling and fluorescence detection[36].(a) Schematic diagram of chip fabrication;(b) diagram of chip structure
Fig. 9 Integrated droplet digital PCR system based on step emulsification[38]
Fig. 10 Capillary-based digital PCR systems[39, 40]
Fig. 11 (a) Digital PCR system based on agarose droplets and primer microspheres[41];(b) digital PCR system based on agarose droplets[42]
Fig. 12 (a) Capillary-based integrated droplet digital PCR system[43];(b) inkjet printing-based integrated online digital PCR system[44]
Fig. 13 (a) Digital PCR system based on surface assisted formation of two-dimensional droplets[45];(b) digital PCR system using microwell arrays for droplet formation[46]
Fig. 14 Digital PCR system based on a SlipChip. (a) The operation schematic of Slipchip[47];(b) Slipchip for multi-volume digital PCRassay[49]
Table 1 Performance comparison of some commercial digital PCR instruments[51]
Classification Instrument Sample
dispersion		 Sample
consumption		 Single sample
reaction unit		 Operating
throughput		 Reaction
unit volume		 Reaction time Detection channel
Micro-
chamber		 ClarityTM digital PCR system Microplate isolation 15 μL 10 000 96 1.5 nL Sample loading
1 min
PCR ~ 2 h		 2
Quant studioTM 3D
digital PCR system		 Microplate isolation 14.5 μL 20 000 24 0.86 nL Sample loading
4 min
PCR ~2.5 h		 2
Constellation digital
PCR system		 Microplate isolation 10 μL 496 96 4 nL Sample loading 12 min
PCR ~1 h		 4
Microfluidic Chip BioMarkTM HD High-throughput genetic
analysis system		 Microvalve 8 μL 765 12 6 nL Sample loading
10 min
PCR ~2 h		 ~5
4 μL 770 48 0.85 nL Sample loading
40 min
PCR ~2 h
Droplet QX200TMMicrodroplet digital PCR system Flow-focusing 18 μL 20000 96 0.85 nL Droplet generation
2 min
PCR ~2 h		 2
RaindropTM digital
PCR system		 Flow-focusing 25~50 μL Up to
1 000 000		 8 5 pL Droplet generation
20~30 min
PCR ~3 h		 2
NaicaTM droplet
digital PCR system		 Step emulsification 20 μL 25 000~
30 000		 4 0.43 nL Droplet generation
15 min
PCR ~1 h		 3
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