数字PCR技术及其在检测领域的应用

doi:10.16288/j.yczz.19-351

遗传 ›› 2020, Vol. 42 ›› Issue (4): 363-373.doi: 10.16288/j.yczz.19-351

数字PCR技术及其在检测领域的应用

冯秀晶, 易红梅, 任星旭, 任佳丽, 葛建镕, 王凤格()

北京市农林科学院玉米研究中心,玉米DNA指纹及分子育种北京市重点实验室,北京 100097

收稿日期:2019-11-18 修回日期:2020-03-12 出版日期:2020-04-20 发布日期:2020-03-31
通讯作者: 王凤格 E-mail:gege0106@163.com
作者简介:冯秀晶,博士,研究方向：转基因植物检测。E-mail: fengxj187@126.com;|易红梅,博士,副研究员,研究方向：玉米分子鉴定。E-mail: yhmmaize@163.com; 冯秀晶和易红梅为并列第一作者。
基金资助:
国家重点研发计划项目编号(2017YFD0102001);转基因生物新品种培育重大专项资助编号(2017ZX08013001)

Digital PCR and its application in biological detection

Xiujing Feng, Hongmei Yi, Xingxu Ren, Jiali Ren, Jianrong Ge, Fengge Wang()

Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Maize Research Center, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, China

Received:2019-11-18 Revised:2020-03-12 Online:2020-04-20 Published:2020-03-31
Contact: Wang Fengge E-mail:gege0106@163.com
Supported by:
Supported by the National Key Research and Development Program of China No(2017YFD0102001);the National Genetically Modified Organisms Key Breeding Projects of China No(2017ZX08013001)

摘要/Abstract

摘要：

随着分子生物学技术的不断发展和需求的多样化,用于核酸检测的各种PCR衍生技术应运而生。数字PCR是一种单分子水平的大规模分区扩增定量核酸检测技术。该技术以微腔室/微孔或微滴作为PCR反应器,无需校准物和绘制标准曲线即可实现对样品初始浓度的绝对定量,具有高灵敏度、高特异性和高精确度的特点。本文详细介绍了数字PCR的技术发展史、作用原理以及仪器平台类型,系统阐述了数字PCR在转基因检测、疾病诊断、环境及食品监管等方面的应用概况,并对该技术的应用前景进行了展望,以期对未来数字PCR的开发利用提供参考。

关键词: 数字PCR, 单分子, 绝对定量

Abstract:

Various derivative technologies based on PCR for nucleic acid detection have emerged with the continuous development and the diverse needs of molecular biology technology. Digital PCR (dPCR) is a nucleic acid detection method for large scale amplification based on a single molecular template, which runs an individual PCR reaction using chambers/wells or droplets. dPCR can be used for absolute quantification for the initial concentration of samples without calibrator and drawing standard curve, showing the characteristics of high sensitivity, specificity, and accuracy. In this review, we introduce the history of technology development, principle, and instrument platform types of digital PCR in detail. Then, we summarize the application of this technology in GMO quantification, disease diagnosis, environment and food supervision. Finally, we describe the application prospect of dPCR, providing a reference for the development and utilization of this technology in the future.

Key words: digital PCR, single molecule, absolute quantification

冯秀晶, 易红梅, 任星旭, 任佳丽, 葛建镕, 王凤格. 数字PCR技术及其在检测领域的应用[J]. 遗传, 2020, 42(4): 363-373.

Xiujing Feng, Hongmei Yi, Xingxu Ren, Jiali Ren, Jianrong Ge, Fengge Wang. Digital PCR and its application in biological detection[J]. Hereditas(Beijing), 2020, 42(4): 363-373.

图/表 3

图1

表1

dPCR仪比较"

类型	供应商	仪器型号	分散成反应单元数目和体积	通量	进样量 (µL)	通道数	特点
微流控芯片数字PCR 仪	美国 Fluidigm	BioMark HD	12 paneL/芯片,765个反应单元/paneL,9180个反应单元/芯片,6 nL /反应单元	12	8	5	全自动微流体控制,可实现12个样品进样和微流体分配,成本高,耗时4 h
	美国 Fluidigm	BioMarkEP1	48个阵列/芯片,770个腔室/阵列,36,960个反应单元/芯片,0.85 nL/反应单元	48	4	5	全自动微流体控制,可实现48个样品进样和微流体分配,成本高,耗时4 h
	美国 ThermoFisher (Life- technlogies)	QuantStudio3D 数字PCR系统	4个板,20,000个反应孔/芯片,0.72 nL/反应单元	24	16	3	全自动,减少移液过程,基于微加工单晶硅芯片,样本之间完全隔离,全程闭管,防止交叉污染,无“死区”;通量低,每次运行只能分析一个样品 , 耗时4 h
	美国 ThermoFisher (Life- technlogies)	OpenArray/ QuantStudio 12K	48个次级阵列/板,64个反应单元/阵列,共计3072个反应单元/板;33 nL/反应单元	4	100	2	全自动芯片处理,全程闭管防止交叉污染,可同时运行4块3072模块,耗时4 h
微滴数字PCR 仪	美国Bio-Rad (QuantaLife)	QX200 ddPCR	微滴生成器8通道设计,可将8个样品分别制备成20,000个液滴,0.837 nL/微滴	1~96	20	2	半自动,常规PCR仪扩增后,利用液滴读取仪读取,操作比较繁琐,配套工具耗材较多,读取耗时较长,防止分散管路堵塞问题
	美国Bio-Rad (RainDance)	Raindrop ddPCR	可将1个样品分割成1000万个微滴,5 pL/微滴	8	25~50	2	原理与QX200类似,设备和芯片耗材成本昂贵,耗时4 h
	中国锐讯生物	DropX-2000	20,000~30,000个微滴, <1 nL/微滴	2~96	20~30	4	人工上样仅需20 s,后续全部自动化,耗时2.5 h
集成微流微孔 PCR仪	法国 Stilla Technologies	Naica™ crystal digital PCR	25,000~30,000个反应单元/芯片,0.43 nL/微滴	12	20	3	集成Sapphire芯片为唯一耗材,人工上样,后续全部自动化,微滴随机平铺成2D阵列,全封闭,无交叉污染,精确到单微滴回溯,耗时2.5 h
集成微流微孔 PCR仪	中国小海龟	BioDigital	20,000余个液滴/芯片, 0.79 nL/微滴	1~96	30	4	芯片式微滴数字PCR,自动分液时间仅需2 min,微滴随机平铺成,无交叉污染,自动读取数据时间为2 min,全封闭,耗时1.5 h

表1

图2

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