数字PCR技术的发展及应用

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。     

液滴微流控系统在数字聚合酶链式反应中的应用研究进展

数字聚合酶链式反应( PCR)技术近年来发展迅速。与以实时荧光定量PCR为代表的传统PCR技术相比,数字PCR技术显著提高了定量分析的精确度和灵敏度。数字PCR的快速发展与近年来微流控技术在数字PCR技术中的广泛应用有着密切的联系。早期的研究和商业化产品使用的是大规模集成流路微流控芯片,加工过程复杂且价格高昂。近年来,液滴微流控芯片被应用到数字PCR技术中,它可以在短时间内产生102~107个微液滴,每一个微液滴都是最多只含有一个目的基因片段的PCR反应器。 PCR扩增后,通过对单个微液滴的观察计数,就可以获得绝对定量的分析数据。本文综述了不同种类的液滴微流控系统在数字PCR技术中的应用,以及液滴数字PCR微流控芯片在生物、医药、环境等领域的应用。     

数字PCR技术进展

数字PCR是近年来迅速发展起来的一种定量分析技术。与传统定量PCR技术不同的是数字PCR不依赖于扩增曲线的循环阈值(C_T)进行定量,不受扩增效率的影响,也不必采用看家基因和标准曲线,具有很好的准确度和重现性, 可以实现绝对定量分析。迄今为止,已有Fluidigm、Bio-Rad等几家公司相继推出了数字PCR产品,这些产品已经在单细胞分析、癌症早期诊断和产前诊断等研究领域显示出巨大的技术优势和应用前景。本文在现有文献基础上,对数字PCR技术的基本原理和定量方法进行介绍,并对该技术的分类及其特点以及目前的主要应用领域进行评述。     

基于非接触电导检测的PCR液滴检测芯片的研究

数字PCR是继实时定量PCR之后新兴发展起来的一种绝对定量分析技术,广泛应用于疾病早期检测诊断和微生物检测分析等领域。然而,在数字PCR的微反应器检测环节,常规的光学检测方法需要复杂的光学设备,应用环境有限,成本高。因此研究了一种基于非接触电导检测的PCR液滴检测芯片,用电导检测取代传统的光学检测,有效降低了数字PCR结果读出系统的成本。通过优化电极结构与排布,采用三电极平行3D电极结构,减小了相对误差,提高了灵敏度,实现了无标记的PCR反应液滴阴性阳性比例检测。在DNA模板浓度为0.5×10~4～3×10~4 copies/μL内实现了良好的相关性(r~2=0.998),检测通量可达100个/秒。     

激光技术在聚合酶链式反应微流控芯片中的应用

评述了激光技术在聚合酶链式反应(polymerase chain reaction,PCR)微流控芯片领域中的应用进展,包括激光技术在PCR微流控芯片微加工、微流体物理参数测量、温度循环控制以及芯片上在线产物检测(包括荧光实时定量/终点和毛细管电泳检测)中的应用。最后,展望了激光技术在未来基于PCR的微全分析系统(micro-total analysis system,μTAS)中的新应用。     

一种快速鉴别食用油的气相色谱指纹法

本发明公开了提供一种实时荧光定量PCR检测HC．MVgBn的分型方法,在PCR反应体系中加入荧光探针,利用荧光信号积累实时监测PCR进程,进行定量定性分析确定HCMVgBn型。本发明封闭反应,无需PCR后处理,避免污染;特异性强,灵敏度高采用对数期分析,摒弃终点数据,定量准确;实时荧光定量PCR技术既可对HCMVgB进行分型,又可对其进行定量;仪器在线式实时检测,结果直观,避免人为判断;可实现一管双检或多检;操作安全,缩短时间,     

聚合酶键式反应生物芯片/微装置的实时定量荧光检测

以微电子机械系统技术而发展起来的聚合酶链式反应(Polymerase Chain Re-action,PCR)生物芯片/微装置,由于具有所需样品和反应混合物体积少,反应速度快以及集成化程度高等优点而日益引起人们的重视。实时定量PCR是利用能特异标记PCR产物的荧光染料来动态显示PCR产物的累积,从而得到PCR扩增曲线的技术。本文介绍了实时定量检测技术及其在PCR生物芯片/微装置中的应用。     

血液乙肝病毒基因检测

全世界每年死于乙型肝炎病毒相关疾病的人数已达60万人,应用荧光定量PCR法检测能够提高乙肝病毒基因的检出率,正确掌握该技术方法能够更加有效地进行疾病诊断和预防工作。基于此,研究针对荧光定量PCR法检测乙肝病毒基因的方法进行探讨。     

数字PCR测定DNA含量及测量结果不确定度评定

数字PCR是一项不依赖校准物的DNA绝对定量技术,将含有DNA模板的反应溶液分配到大量独立的微室中并且进行扩增反应,通过统计反应室中的阳性信号来定量DNA的拷贝数。采用数字PCR技术对噬菌体λDNA的含量进行测定,并对测定结果进行不确定度评定。结果显示,由数字PCR测得的λDNA的含量为(2.43±0.35)μg/管(k=2),该结果与同位素稀释质谱法测量结果相吻合。     

智能数字聚合酶链式反应系统的开发与应用验证

数字聚合酶链式反应(digital polymerase chain reaction,dPCR)技术可以针对低浓度的目标核酸分子实现精确的绝对定量检测,在各类疾病的检测与治疗方面有着极大应用价值. 针对目前商业数字PCR仪造价昂贵、体积庞大等缺点,基于智能手机与微流控芯片,设计开发了一种低成本、高集成的智能数字PCR设备. 介绍了硬件系统的制作以及整机的整合搭建过程. 采用PID算法,结合温控电路与半导体制冷片等硬件,进行了PCR温度循环的精准控制. 最后,采用自适应阈值分割法对采集到的荧光图像进行了处理,并依据泊松分布的规律对统计结果进行了校正,完成了对PCR反应后采集到荧光图像的结果分析与检测.     

Optimization and Verification of Droplet Digital PCR Even-Specific Methods for the Quantification of GM Maize DAS1507 and NK603

In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on “Method Verification.” (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed.     

Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction

Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in real-time PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.     

Simultaneous estimation of detection sensitivity and absolute copy number from digital PCR serial dilution

Digital polymerase chain reaction (dPCR) is a refinement of the conventional PCR approach to nucleic acid detection and absolute quantification. Digital PCR works by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions. Current quantification methods rely on the assumption that the PCR reactions are always able to detect single target molecules. When the assumption does not hold, the copy numbers will be severely underestimated. We developed a novel dPCR quantification method which determines whether the single copy assumption is violated or not by simultaneously estimating the assay sensitivity and the copy numbers using serial dilution data sets. The implemented method is available as an R package “digitalPCR”.     

Compressed Air-Driven Continuous-Flow Thermocycled Digital PCR for HBV Diagnosis in Clinical-Level Serum Sample Based on Single Hot Plate

We report a novel compressed air-driven continuous-flow digital PCR (dPCR) system based on a 3D microfluidic chip and self-developed software system to realize real-time monitoring. The system can ensure the steady transmission of droplets in long tubing without an external power source and generate stable droplets of suitable size for dPCR by two needles and a narrowed Teflon tube. The stable thermal cycle required by dPCR can be achieved by using only one constant temperature heater. In addition, our system has realized the real-time detection of droplet fluorescence in each thermal cycle, which makes up for the drawbacks of the end-point detection method used in traditional continuous-flow dPCR. This continuous-flow digital PCR by the compressed air-driven method can meet the requirements of droplet thermal cycle and diagnosis in a clinical-level serum sample. Comparing the detection results of clinical samples (hepatitis B virus serum) with commercial instruments (CFX Connect; Bio Rad, Hercules, CA, USA), the linear correlation reached 0.9995. Because the system greatly simplified the traditional dPCR process, this system is stable and user-friendly.     

Addition of gold nanoparticles to real-time PCR: effect on PCR profile and SYBR Green I fluorescence

Real-time quantitative polymerase chain reaction (qPCR) is the industry standard technique for the quantitative analysis of nucleic acids due to its unmatched sensitivity and specificity. Optimisation and improvements of this fundamental technique over the past decade have largely consisted of attempts to allow faster and more accurate ramping between critical temperatures by improving assay reagents and the thermal geometry of the PCR chamber. Small gold nanoparticles (Au-NPs) have been reported to improve PCR yield under fast cycling conditions. In this study, we investigated the effect of Au-NPs on optimised real-time qPCR assays by amplifying DNA sequences from genetically modified canola in the presence and absence of 0.9 nM Au-NPs of diameter 12 ± 2nm. Contrary to expectations, we found that Au-NPs altered the PCR amplification profile when using a SYBR Green I detection system due to fluorescence quenching; furthermore, high-resolution melt (HRM) analysis demonstrated that Au-NPs destabilised the double-stranded PCR product. The results indicate that effects on the assay detection system must be carefully evaluated before Au-NPs are included in any qPCR assay. Figure Raw amplification profiles in the presence and absence of gold nanoparticles     

Methods for detection of GMOs in food and feed

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.     

Advances in molecular techniques for the detection and quantification of genetically modified organisms

Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.     

GMO quantification: valuable experience and insights for the future

Cultivation and marketing of genetically modified organisms (GMOs) have been unevenly adopted worldwide. To facilitate international trade and to provide information to consumers, labelling requirements have been set up in many countries. Quantitative real-time polymerase chain reaction (qPCR) is currently the method of choice for detection, identification and quantification of GMOs. This has been critically assessed and the requirements for the method performance have been set. Nevertheless, there are challenges that should still be highlighted, such as measuring the quantity and quality of DNA, and determining the qPCR efficiency, possible sequence mismatches, characteristics of taxon-specific genes and appropriate units of measurement, as these remain potential sources of measurement uncertainty. To overcome these problems and to cope with the continuous increase in the number and variety of GMOs, new approaches are needed. Statistical strategies of quantification have already been proposed and expanded with the development of digital PCR. The first attempts have been made to use new generation sequencing also for quantitative purposes, although accurate quantification of the contents of GMOs using this technology is still a challenge for the future, and especially for mixed samples. New approaches are needed also for the quantification of stacks, and for potential quantification of organisms produced by new plant breeding techniques.