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”.     

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.     

Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding samples

Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to next-generation sequencing. End point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2,000-fold differences in DNA quantity after amplification. Next-generation sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of qPCR amplification.     

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。     

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.     

Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR

Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that allows quantitative amplification detection at higher multiplexing by the integration of amplification in solution and monitoring via hybridization to a microarray in real-time. This method does not require any manipulation of the PCR product and runs in a single closed chamber. Employing labeled primers, one of the main challenges is to measure surface signals against a high fluorescence background from solution. A compact, confocal scanner is employed, based on miniaturized optics from DVD technology and combined with a flat thermocycler for simultaneous scanning and heating. The feasibility of this method is demonstrated in singleplex with an analytical sensitivity comparable to routine qrtPCR.     

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.     

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     

Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.     

A multiplex, bead-based array for profiling plant-derived components in complex food matrixes

Authentication of processed food ingredients is becoming an important issue for customers, and some DNA-based analytical methods have been developed, especially for animal products. As food products typically contain several different ingredients, a current challenge is to increase the multiplexing capacity of DNA-based methods, to develop “all-in-one” assays. Oligonucleotide-coupled, bead-based suspension arrays are sensitive and reproducible multiplex analytical tools. We applied the Multi-Analyte Profile (xMAP?) technology to develop an assay able to concurrently detect five different plant components in mixed flours and in processed feed and food. Capture probes were targeted to species-specific DNA polymorphisms present within the first intron of plant β-tubulin genes, which can be amplified by the tubulin-based polymorphism-amplification method (TBP-PCR). The workflow is very simple and straightforward, consisting of a PCR amplification step with universal primers, followed by the direct hybridization assay. Results are highly reproducible. For each single plant species, the absolute detection limit was as low as one target DNA copy. In complex mixtures of flours derived from seeds or from commercial dry “pasta,” relative limits of detection ranged, in weight, from 2 % for soybean to less than 0.5 % for wheat. The specificity of the capture probes and the high sensitivity of the method allowed the successful determination of the analytical composition of three feeds as well as eleven food samples, such as snacks, biscuits, and pasta. The multiplexing ability of the assay (up to 100 different analytes) provides scalability and flexibility, in response to specific needs. Schematic representation of the four-step assay for the determination of plant-derived components in food samples

Tuning single nucleotide discrimination in polymerase chain reactions (PCRs): synthesis of primer probes bearing polar 4'-C-modifications and their application in allele-specific PCR

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.     

Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue

Extensive collections of formalin-fixed paraffin-embedded (FFPE) tissues exist that could be exploited for genetic analyses in order to provide important insights into the genetic basis of disease or host/pathogen cointeractions. We report here an evaluation of a 44 SNP multiplex genotyping method, multiplex PCR with minisequencing (MPMS), on 92 DNA extractions performed on six archival FFPE samples of variable DNA quality, which date between 9 and 25 years old. On the three extracts with highest quality, we found the assay efficiency to be near 100%. However, the efficiency of the lowest quality extracts varied significantly. In this study, we demonstrate that although direct measures of DNA concentration in the extracts provide no useful information with regard to subsequent MPMS success, the success of the assay can be determined to some degree a priori, through initial screening of the DNA quality using a simple quantitative real-time PCR (qPCR) assay for nuclear DNA, and/or an assay of the maximum PCR amplifiable size of nuclear DNA. MPMS promises to be of significant use in future genetic studies on FFPE material. It provides a streamlined approach for retrieving a large amount of genetic information using simple, single reactions and minute amounts of archival tissue/DNA. In the light of this evidence, we suggest that the systematic screening of FFPE collections may in the future provide valuable insights into the past.     

High-throughput single molecule screening of DNA and proteins

We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification.     

Strategy for high-fidelity multiplex DNA copy number assay system using capillary electrophoresis devices

Structural variation of human genome such as duplications and deletions, collectively termed copy number variation (CNV), is one of the major genetic variations. Reliable and efficient measurement of CNV will be essential to develop diagnostic tools for CNV-related diseases. We established a strategy based on multiplex PCR and capillary electrophoresis (CE) for reliable CNV assay. Multiplex-PCR was performed using five primer sets for target loci and a diploid control (DC). We designed primers satisfying three conditions: different size of each PCR product for CE separation, unified annealing temperature for multiplex PCR, and suitability for quantitative PCR (qPCR). We defined the accurate PCR cycles for quantification of copy numbers at which the amplifications for all targets were supposed to be exponential, named maximum doubling cycle. CE was carried out with PCR product and the ratio of the peak areas (target/diploid control) was calculated. Our multiplex PCR-CE analysis reliably determined copy numbers of X chromosome with variable copies ranging from 1 to 5 and showed higher reliability than qPCR (correlation coefficient 0.996 versus 0.898). When measuring the six randomly selected autosomal CNV targets using our multiplex PCR-CE, the results agreed with those from qPCR. In addition, our strategy was validated for the broad application to commonly used CE devices. Taken together, this assay will be useful for accurate analysis of multiple disease-associated CNVs in a clinical setting.     

High-performance closed-tube PCR based on switchable luminescence probes

We introduce a switchable lanthanide luminescence reporter technology based closed-tube PCR for the detection of specific target DNA sequence. In the switchable lanthanide chelate complementation based reporter technology hybridization of two nonfluorescent oligonucleotide probes to the adjacent positions of the complementary strand leads to the formation of a highly fluorescent lanthanide chelate complex. The complex is self-assembled from a nonfluorescent lanthanide chelate and a light-harvesting antenna ligand when the reporter molecules are brought into close proximity by the oligonucleotide probes. Outstanding signal-to-background discrimination in real-time PCR assay was achieved due to the very low background fluorescence level and high specific signal generation. High sensitivity of the reporter technology allows the detection of a lower concentration of amplified DNA in the real-time PCR, resulting in detection of the target at the earlier amplification cycle compared to commonly used methods.     

A simple and precise diagnostic method for spinal muscular atrophy using a quantitative SNP analysis system

A simple and precise diagnostic method for spinal muscular atrophy (SMA) using high‐resolution CE‐based single‐strand conformation polymorphism (CE‐SSCP) was developed in this study. SMA is a common genetic disorder caused by an abnormality in the relative copy numbers of SMN1 and its centromeric copy SMN2, which differ only in two nucleotides, namely at exons 7 and 8. Therefore, the precise discrimination of the differences in sequence as well as their relative quantities is crucial for the diagnosis of SMA. Multiplex ligation‐dependent probe amplification and sequence‐sensitive DNA separation using hydroxyethyl cellulose and hydroxypropyl cellulose blended polymer matrix are currently the available methods used in the diagnosis of SMA. However, these methods are limited by their extended hybridization step and low resolution. In this study, the simultaneous discrimination of SMN exons 7 and 8 was successfully demonstrated using high‐resolution CE‐SSCP. Unlike the previously reported alternative method, single base differing amplicons were baseline‐separated because of its extraordinary resolution, thus providing accurate and precise quantification of each paralog.     

Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits

Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.