数字PCR在食品安全检测中的应用研究进展

聚合酶链反应(PCR)在动植物源掺杂鉴别、转基因成分和致病微生物等食品安全检测领域成为日趋重要的检测技术。从传统PCR、荧光PCR到数字PCR,PCR技术逐渐从定性分析、半定量分析发展到准确定量,不仅提升了准确度和检测效率,同时也扩展了食品检测范围,使食品安全的监管更加精细化。该文总结了近5年来数字PCR在食品安全检测中的研究进展,比较了传统PCR、荧光定量PCR和数字PCR的相关标准制订情况,列举、讨论了数字PCR在不同食品安全领域检测中的技术进展和存在的问题,并对数字PCR未来发展方向进行了展望。     

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。     

利用实时荧光PCR鉴定小体鲟物种的快速方法

从小体鲟线粒体基因筛选出位于COⅠ(细胞色素氧化酶)基因中的一段保守序列,针对其设计引物,优化SYBR Green实时荧光PCR反应体系,建立了一种鉴定小体鲟的实时荧光PCR定性检测方法.该方法检测小体鲟DNA灵敏度为0.04mg/L.通过对采集和市售小体鲟鱼样品检测,该方法可检测出样品中的小体鲟成分.实验证明该方法可对血液样品和组织样品中的小体鲟进行种源鉴别.     

病理切片疫苗安全性指标自动检测技术EI北大核心CSCD

针对猪病灭活疫苗研发、生产过程中和疫苗病理安全性检测时,存在对油乳佐剂残留、炎症细胞聚集2大主要安全性指标评估人工统计不准确、效率低的问题,建立了一套小型病理切片的病理图像采集显微系统,并提出了针对疫苗安全性指标的定量分析算法,实现了对病理切片快速数字化图像采集,以及对待测指标的定量统计分析及检测结果的可视化标记。搭建了一套高分辨率病理图像采集显微系统,并建立了炎症细胞聚集区域病理图像分割模型和油乳佐剂疫苗残留区域病理图像分割模型。对整张数字病理图像进行窗口扫描,得到整张病理图像2大指标的分析结果并进行可视化视觉标注。结果表明,此检测系统能在数秒内完成病理切片的病理图像采集,并对关键疫苗病理安全性检测指标进行可视化标记和定量分析,对油佐乳剂残留像素检出覆盖率误差为0.015,检测像素交并比为0.89,炎症细胞聚集平均像素检出覆盖率误差为0.01,检测像素交并比为0.74,实现了对病理安全性指标高精度无干预的自动检测分析和标记,可以满足实际生产和研究的检测需求。     

转基因玉米的多重PCR-毛细管电泳-激光诱导荧光检测方法研究

采用三重PCR反应, 同时扩增CaMV 35S启动子、 hsp70 intron1和CryIA(b)基因之间序列以及Invertase基因, 扩增产物用无胶筛分毛细管电泳-激光诱导荧光检测, 从而建立了多重PCR-毛细管电泳-激光诱导荧光快速检测转基因玉米的新方法. 对影响多重PCR扩增和毛细管电泳的因素进行了优化. 在优化的条件下, 本方法可以同时检测转基因玉米样品中3种外源基因. 经序列测试证实, 三重PCR 扩增产物的序列与原基因完全一致, 表明扩增结果可靠. 该方法能检出0.05% MON810转基因玉米成分, 远低于欧盟对转基因食品规定标识的质量分数阈值(1％). 该方法对玉米及其制品的检测结果与实时荧光PCR方法的检测结果一致, 与传统的琼脂糖凝胶电泳法相比, 具有特异性高\, 快速及灵敏等优点, 适用于玉米中转基因成分以及转基因玉米MON810品系的快速筛选、 鉴定和检测, 能满足我国实施转基因食品标签法规的要求.     

巴西果仁致敏原的实时荧光PCR检测

针对巴西果仁2Salbumin mRNA基因设计引物、探针,在实时荧光PCR仪上进行扩增、检测和结果分析.结果显示:该组探针和引物对巴西果仁有很强的特异性,除巴西果仁外,其余8种对照树果材料均未检测到荧光信号,巴西果仁成分检测灵敏度达到0.1%.该方法具有灵敏度高、快速、简便的特点,可用于巴西果仁致敏原成分的定量检测.     

透射电子显微镜侧插式CCD相机数字图片放大倍数的解析

针对数字化透射电子显微镜与传统透射电子显微镜在观察记录系统上存在的差异,通过比较分析数字化透射电子显微镜侧插式电荷耦合器件(CCD)相机采集的图像、操作界面View视窗观察到的图像与电镜示数放大倍数之间的差异,详细地分析了数字化透射电子显微镜侧插式CCD相机采集的数字图片的放大倍数发生变化的原因.可以帮助应用电镜的广大教师及科研工作者更加快速、直观地掌握数字图片确切的放大倍数,更有利于结果的分析.     

两种Taqman探针的制备及荧光检测性能比较

采用固相合成法,分别合成FAM/TAMRA和FAM/BHQ2双荧光标记的Taqman探针,得到的粗产物分别采用乙醇沉淀法、离子对反相色谱法、凝胶过滤柱脱盐法纯化.采用实时荧光PCR法,分别从感染的南瓜果实中检测到黄瓜绿斑驳花叶病毒(CGMMV),扩增曲线表明采用该实验条件制备的探针检测性能良好.在相同荧光PCR反应体系...     

实时荧光等位基因特异性扩增法快速检测K-ras癌基因点突变

将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification, ASA)方法相结合, 发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法. 将该法用于检测K-ras癌基因第12位密码子发生的点突变, 分别采用针对其不同点突变方式(GAT, GTT, CGT)设计的突变型引物对待测样品进行ASA, 只有突变型样品能被顺利扩增出双链DNA产物, 该产物才能与双链DNA染料SYBR Green Ⅰ结合, 发出荧光信号从而被检测到. 用该法检测31例结肠癌组织中的K-ras癌基因点突变, 其中有15例样品检出为突变型. Real-time ASA法可检测到样品中含量为1/1000的突变型基因, 具有灵敏、快速、简便、安全、高通量和低成本等优点, 可望用于大量临床样本的点突变筛查.     

复合式蝎形引物实时定量检测端粒酶延伸产物

针对端粒酶延伸产物中靶基因序列的特殊性,开发了一种可产生荧光的复合式蝎形引物,该引物的5'端带有可特异性检测靶基因的探针序列,PCR阻断剂将其与引物序列连接.当复合式蝎形引物延伸,探针序列与同一分子内的靶基因杂交,荧光信号产生.运用该技术,建立了定量检测端粒酶延伸产物的实时荧光PCR方法.该法可在快速PCR循环条件下,对0.15～1.50×10³ amol/μL范围内的样品进行定量检测,线性相关系数R²=0.9992.该法操作简便,无需PCR后额外的检测步骤.     

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.     

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”.     

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.     

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.     

Effect of sustained elevated temperature prior to amplification on template copy number estimation using digital polymerase chain reaction

Digital polymerase chain reaction (dPCR) has the potential to enable accurate quantification of target DNA copy number provided that all target DNA molecules are successfully amplified. Following duplex dPCR analysis from a linear DNA target sequence that contains single copies of two independent template sequences, we have observed that amplification of both templates in a single partition does not always occur. To investigate this finding, we heated the target DNA solution to 95 °C for increasing time intervals and then immediately chilled on ice prior to preparing the dPCR mix. We observed an exponential decline in estimated copy number (R(2)≥ 0.98) of the two template sequences when amplified from either a linearized plasmid or a 388 base pair (bp) amplicon containing the same two template sequences. The distribution of amplifiable templates and the final concentration (copies per μL) were both affected by heat treatment of the samples at 95 °C from 0 s to 30 min. The proportion of target sequences from which only one of the two templates was amplified in a single partition (either 1507 or hmg only) increased over time, while the proportion of target sequences where both templates were amplified (1507 and hmg) in each individual partition declined rapidly from 94% to 52% (plasmid) and 88% to 31% (388 bp amplicon) suggesting an increase in number of targets from which both templates no longer amplify. A 10 min incubation at 95 °C reduced the initial amplifiable template concentration of the plasmid and the 388 bp amplicon by 59% and 91%, respectively. To determine if a similar decrease in amplifiable target occurs during the default pre-activation step of typical PCR amplification protocol, we used mastermixes with a 20 s or 10 min hot-start. The choice of mastermix and consequent pre-activation time did not affect the estimated plasmid concentration. Therefore, we conclude that prolonged exposure of this DNA template to elevated temperatures could lead to significant bias in dPCR measurements. However, care must be taken when designing PCR and non-PCR based experiments by reducing exposure of the DNA template to sustained elevated temperatures in order to improve accuracy in copy number estimation and concentration determination.     

Lambda genomic DNA quantification using ultrasonic treatment followed by liquid chromatography–isotope dilution mass spectrometry

Quantification of genomic DNA that is traceable to the SI was performed successfully by measuring the individual nucleotides. Specifically, ultrasound was used to shear lambda genomic DNA into fragments of less than 200 base pairs, followed by deoxyribonuclease Ι and phosphodiesterase Ι digestion and liquid chromatography–isotope dilution mass spectrometry (LC-IDMS) quantification to estimate the mass fraction of the lambda DNA, based on the constituent deoxynucleotide monophosphates (dNMPs) within the molecule. Digital PCR (dPCR) was employed to quantify the same lambda DNA solution to provide independent data for comparing the performance of two quantitative methods. On the basis of the LC-IDMS measurement after ultrasonic treatment of the sample, the concentration of lambda DNA was 273.1 ± 9.8 μg/g (expanded uncertainty at the 95% confidence interval). This shows good agreement with the data from dPCR. Additionally, the result calculated on the basis of the sum of the concentrations of the four dNMPs is the same as that calculated on the basis of the sequence, which indicates that knowledge of the DNA sequence and length is unnecessary to measure the total DNA concentration when applying ultrasonic treatment–LC-IDMS.     

An ultra-sensitive DNA assay based on single-molecule detection coupled with hybridization accumulation and its application

An ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation was developed. In this assay, target DNA (tDNA) in solution was accumulated on a silanized substrate blocked with ethanolamine and bovine serum albumin (BSA) through a hybridization reaction between tDNA and capture DNA immobilized on the substrate. The tDNA on the substrate was labeled with quantum dots which had been modified with detection DNA and blocked with BSA. The fluorescence image of single QD-labeled tDNA molecules on the substrate was acquired using total internal reflection fluorescence microscopy. The tDNA was quantified by counting the bright dots on the image from the QDs. The limit of detection of the DNA assay was as low as 6.4 × 10(-18) mol L(-1). Due to the ultra-high sensitivity, the DNA assay was applied to measure the beta-2-microglobulin messenger RNA level in single human breast cancer cells without a need for PCR amplification.