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化学进展 2020, Vol. 32 Issue (5): 581-593 DOI: 10.7536/PC190913 前一篇   后一篇

• 综述 •

数字PCR技术的发展及应用

李慧调1, 潘建章1,**(), 方群1,**()   

  1. 浙江大学化学系 微分析系统研究所 杭州 310058
  • 收稿日期:2019-09-09 修回日期:2019-12-06 出版日期:2020-05-15 发布日期:2020-02-20
  • 通讯作者: 潘建章, 方群
  • 基金资助:
    国家自然科学基金项目(21974122); 国家自然科学基金项目(21827806); 国家自然科学基金项目(21435004)
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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:2019-09-09 Revised:2019-12-06 Online:2020-05-15 Published:2020-02-20
  • 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)

数字PCR(Digital PCR, dPCR)是继实时荧光定量PCR(Real-time quantitative PCR, qPCR)之后发展的高灵敏核酸绝对定量分析技术,通过把反应体系均分到大量独立的微反应单元中进行PCR扩增,并根据泊松分布和阳性比例来计算核酸拷贝数实现定量分析。与传统PCR技术相比,数字PCR 技术不依赖于标准曲线,具有更高灵敏度、准确度及高耐受性,可实现对样品的绝对定量分析。近年来,随着微流控技术日臻成熟,基于微流控技术的数字PCR技术得到了快速的发展,在基因突变检测、拷贝数变异检测、病毒微生物检测、转基因食品检测以及测序等方面均得到广泛的应用。本文对数字PCR的原理、技术发展和应用进行了概述。

关键词： 微流控技术, 数字PCR, 绝对定量

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
()
图1 数字PCR分析原理[8]
Fig. 1 The principle of Digital PCR assay[8]
图2 Web of Science数据库统计1999~2018年间数字PCR 论文发表情况
Fig. 2 Numbers of published articles in the field of digital PCR from 1999 to 2018 in Web of Science
图3 基于通孔阵列的数字PCR芯片 (a)不锈钢数字PCR芯片[14];(b)QuantstudioTM 3D数字PCR芯片[15];(c)ClarityTM数字PCR芯片[16];(d)Constellation数字PCR芯片[17]
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]
图4 (a)具有PDMS微阀结构的数字PCR芯片及核酸扩增结果[19];(b)具有飞升级体积微反应室的数字PCR芯片[20]
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]
图5 (a)具有百万个皮升级微反应室的数字PCR芯片[21];(b)自分散数字PCR芯片[23];(c)自吸分液式数字PCR芯片[24];(d)PET旋转圆盘数字PCR芯片[25]
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]
图6 BEAMing技术原理示意图[28]
Fig. 6 Schematic diagram of BEAMing technique[28]
图7 集成液滴生成、液滴重注入、电致液滴融合和在线荧光检测的微流控芯片数字PCR分析系统[33,34]
Fig. 7 Digital PCR analysis system integrated droplet formation, droplet reinjection, electron droplet fusion, and online fluorescence detection[33, 34]
图8 集成液滴生成、分裂、排布、热循环和荧光检测等过程的数字PCR系统[36]:(a) 芯片加工示意图;(b) 芯片结构图
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
图9 基于阶梯乳化的集成液滴数字PCR系统[38]
Fig. 9 Integrated droplet digital PCR system based on step emulsification[38]
图10 基于毛细管的数字PCR系统[39, 40]
Fig. 10 Capillary-based digital PCR systems[39, 40]
图11 (a)基于琼脂糖液滴和引物微球的数字PCR系统[41];(b)基于琼脂糖液滴的数字PCR系统[42]
Fig. 11 (a) Digital PCR system based on agarose droplets and primer microspheres[41];(b) digital PCR system based on agarose droplets[42]
图12 (a)基于毛细管的集成液滴数字PCR系统[43];(b)基于喷墨打印技术的在线检测的数字PCR系统[44]
Fig. 12 (a) Capillary-based integrated droplet digital PCR system[43];(b) inkjet printing-based integrated online digital PCR system[44]
图13 (a)基于表面辅助形成二维液滴阵列的数字PCR系统[45];(b)基于微孔阵列形成液滴的数字PCR系统[46]
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]
图14 基于滑动芯片的数字PCR系统 (a)滑动芯片操作示意图[47];(b)用于多体积数字PCR分析的滑动芯片[49]
Fig. 14 Digital PCR system based on a SlipChip. (a) The operation schematic of Slipchip[47];(b) Slipchip for multi-volume digital PCRassay[49]
表1 部分商品化数字PCR仪器性能对比[51]
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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摘要

数字PCR技术的发展及应用