Research Article: Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

Date Published: March 24, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Tigst Demeke, David Dobnik.

http://doi.org/10.1007/s00216-018-1010-1

Abstract

The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing.

Partial Text

Genetically modified organisms (GMOs) have already passed a two-decade milestone of presence on the market, and the number of GMOs as well as the worldwide area planted with biotech crops continues to increase steadily [1]. Many countries regulate the cultivation and trade of GMOs [2, 3], whereas others have at least some kind of authorization system and/or labelling requirements in place for GMOs present in food and feed chains [3, 4]. Labelling thresholds are relatively low in some countries; for example, in the European Union (EU) Regulation (EC) 1829/2003 [5] has set the labelling threshold for food products that contain, consist of or are produced from authorized GMOs at 0.9%. For feed samples there is an even lower threshold of 0.1% for GMOs, for which authorization is either pending or has expired (so-called low-level presence) [6]. Additionally, there is zero tolerance for unapproved GMOs in EU countries and some other countries. As a consequence, sensitive and accurate methods must be used for GMO detection and quantification to check product labelling compliance with the legislation. During the last 20 years, several approaches have been developed for GMO detection and quantification, and these are generally divided into two groups: protein-based and DNA-based methods. Because of several performance parameters, DNA-based methods are more widely accepted and used. One of the most promising DNA-based methods is digital polymerase chain reaction (dPCR), which is critically reviewed in this article.

In comparison with conventional end-point PCR and qPCR, dPCR has a number of advantages. The biggest advantage is the capacity of dPCR for absolute quantification of a target without reference to a standard/calibration curve. This minimizes the effect of matrix differences between the calibrant and the test sample, which could cause different amplification efficiencies [49–51]. Because of the principle of high-level sample partitioning, the results obtained with dPCR are very precise [50, 51] and accurate even at very low target copy numbers [52]. Sample partitioning also allows reliable detection of rare targets in a high background of non-target DNA, which is important for GMO analysis, where a transgene (GMO event) might be present at much lower concentrations than the reference gene (endogene). Another important advantage of dPCR is its lower sensitivity to PCR inhibitors. Finally, an important aspect of routine analyses is cost-efficiency. Although analysis of GMO samples by simplex dPCR is more expensive, the use of multiplex approaches moves the scales in favour of dPCR [53, 54].

Several dPCR platforms are available (Table 1) and generally they can be divided in two groups: droplet dPCR (ddPCR; emulsion based) and chip-based dPCR (cdPCR; microfluidic) [55]. For two ddPCR platforms (Bio-Rad’s QX100/QX200 and RainDance’s RainDrop) the reaction mixture is divided into several individual droplets (thousands to millions). Each droplet is amplified by PCR cycling, and amplified droplets are transferred to the droplet reading instrument to determine the number of positive and negative droplets. The RainDrop system provides higher sensitivity (can detect very low concentrations), with millions of droplets generated per sample. The QX100/QX200 and RainDrop platforms are widely used for absolute quantification of GMOs [49, 53, 54, 56, 57, 69, 77]. The QX100 and QX200 systems create around 20,000 droplets per well and have a relatively high throughput (96-well plates are used) compared with the RainDrop platform, which allows analysis of only eight reactions at a time, which are in turn divided into millions of droplets.Table 1Examples of digital polymerase chain reaction platforms availableBioMark HD/EP1 (Fluidigm)QuantStudio 3D (Life Technologies)Constellation/Constellation modules (Formulatrix)Clarity (JN Medsys)Naica (Stilla Technologies)RainDrop (RainDance Technologies)QX200 (Bio-Rad)Partitions765 or 77020,0008000 or 32,00010,00030,0005 × 106–10 × 10620,000Total reaction volume (μL)4–814.510152025–5020Samples per run12 or 482496 or 243212896Duration (h)~4~3~1.5~4~2~7–8~6DyesFAM/EvaGreen, VIC, ROX (Cy5)FAM/SYBR, VIC, ROX5 (FAM/EvaGreen, HEX, ROX, NED, TED, Cy5) for Constellation or 8 different wavelenghts for Constellation ModuleFAM/SYBR/EvaGreen, VIC/HEXFAM, Cy3/VIC/HEX, Cy5FAM, VICFAM/EvaGreen, VIC/HEXMaster mixOpenProprietaryOpenOpenProprietaryOpenProprietary

DNA quality is a key factor for successful PCR. The type of samples used, the DNA extraction methods, etc. can affect the quality of extracted DNA (e.g. presence/absence of inhibitors), which can have an impact on amplification with PCR [63]. Demeke et al. [64] reported comparison of seven DNA extraction kits with a cetyltrimethylammonium bromide (CTAB) method for three different genetically modified ingredients: canola, flax and soybean. The extracted DNA was tested with qPCR and the RainDrop ddPCR system. The RainDrop ddPCR system gave more variable results than qPCR. Most of the kits were appropriate for both ddPCR and qPCR for canola and soybean samples, but only one of the seven DNA extraction kits produced consistent results with RainDrop ddPCR for flax samples (Table 2). Canola, flax and soybean DNAs extracted with the CTAB method and purified with a DNA Clean & Concentrator-25 kit were suitable for both RainDrop ddPCR and qPCR assays [64].Table 2Suitability of DNA extraction kits for quantitative polymerase chain reaction (qPCR) and RainDrop droplet digital polymerase chain reaction (dPCR) for different GM seed samplesDNA extraction methodCanolaFlaxSoybeanFast ID DNA extraction kit✓✓✓FastDNA Spin kit✓qPCR only✓GM Quicker 2 kit✓NA✓OmniPrep for plant kitNAqPCR onlyqPCR onlyNucleoSpin Food kit✓qPCR only✓Plant DNAzol reagentNANANADNeasy mericon Food kit✓ND✓CTAB✓✓✓Compiled from Demeke et al. [64]. Cetyltrimethylammonium bromide (CTAB)-extracted DNA was purified with a DNA Clean & Concentrator kitNA data not available because DNA extraction was not successful, ND not determined (the DNA yield was low and not sufficient for polymerase chain reaction), tick worked for both dPCR and qPCR. CTAB extracted DNA was purified with DNA Clean & Concentrator kit

The amount of DNA used for dPCR can differ according to the instrument used and the sensitivity required. For example, up to 1000 ng DNA can be used for RainDrop ddPCR to detect a low concentration of genetically modified materials [69], and 100 ng DNA has been used for the QX100/QX200 system and other systems. Generally, the same amount of DNA used for real-time qPCR can also be used for dPCR. At NIB, DNA quantity is usually assessed by means of a preliminary qPCR run targeting plant endogenes. Our experience has shown that spectroscopic measurement is not accurate enough. From comparison of measurements with NanoDrop, Qubit and ddPCR, it was observed that NanoDrop overestimated the quantity of genomic DNA by more than two times and Qubit overestimated it by around 50% when compared with the ddPCR results. For cdPCR (e.g. Fluidigm), the assessment of DNA quantity in a reaction is more problematic than in ddPCR because of the narrow dynamic range. Independent of the quantification method, it is important to ensure there are non-denaturing conditions for DNA samples because the quantification result for double-stranded DNA can differ from that for single-stranded DNA by 100%.

In principle, as already mentioned, the absolute target concentration in a sample is calculated on the basis of number of positive partitions and all accepted partitions and by use of a Poisson distribution [38]. The final absolute target concentration in a sample is calculated according to Eq. 1:1documentclass[12pt]{minimal}
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begin{document}$$ {T}_{mathrm{c}}=-ln left(1-frac{P}{R}right)times left(frac{1}{V_{mathrm{d}}}right)times D, $$end{document}Tc=−ln1−PR×1Vd×D,where Tc is the mean target concentration (copies per microlitre), P is the number of positive partitions, and R is the number of partitions analysed. As can be seen from Eq. 1, the target concentration also depends on the partition volume (Vd). A dilution factor for the original sample before PCR (D) is also considered in the equation. Essentially, three factors affect the final result: (1) correct classification of partitions as positive, (2) correctly determined partition volume and (3) a dilution factor.

Multiplexing is readily available in dPCR systems, as all platforms include filters that allow detection of fluorescence in at least two channels (FAM and HEX/VIC). Some have the option of even higher multiplexing, because of the availability of filters for up to five fluorescence channels (Table 1). Duplex absolute quantification is very suitable for GMO analysis, as transgenes and endogenes can be quantified in the same reaction, and thus it can be easily implemented into the testing scheme. Morisset et al. [49] reported on the suitability of duplex reaction for quantification of MON810 transgenic maize. To test the transferability of such a protocol to other laboratories, one DNA sample was tested in three independent laboratories in the Decathlon project (http://www.decathlon-project.eu). The results showed good comparability of determined values between laboratories in terms of absolute copy numbers determined for each target and GMO content (Table 3).Table 3Absolute copy numbers for stock DNA for MON810 and hmgA target determined by three independent laboratories on the same DNA sample by duplex droplet digital polymerase chain reaction and calculated genetically modified (GM) contentTargetLaboratory 1Laboratory 2Laboratory 3Coefficient of variation (%)MON8102768277529519228224092599hmgA76,87471,21380,6161158,56662,66576,312GM content (%)3.73 ± 0.213.87 ± 0.253.54 ± 0.224.5The results for two dilutions, each tested in duplicate, are presented for each target. The results for GM content are presented as an average from all replicates together with the 95% confidence interval

Digital PCR (dPCR) is being used for a wide range of applications in medical, environmental and agricultural areas. The most obvious advantage of dPCR is the possibility to obtain accurate absolute target concentration with no standard curve requirements. Selection of the proper dPCR instrument for a particular laboratory’s need is important. Currently available dPCR instruments seem to be suitable for quantification of GMO events. However, there is variation in throughput and sensitivity, and thus laboratories should assess their needs and budget before making a final decision. Assays used for qPCR can readily be transferred to dPCR; nevertheless, some optimization of the primer and probe concentrations might potentially improve the overall assay performance. A more thorough evaluation and/or verification is essential, especially for assays used in a multiplex dPCR format. Many reports have shown that dPCR is less sensitive to inhibitors compared with qPCR, indicating that it might be a method of choice for samples where the presence of inhibitors is expected. It has also been reported that the DNA extraction method used and DNA quality affect dPCR results. Care must also be taken with partition or droplet volumes assigned by the manufacturer, as the actual values could differ and adjustments may be necessary. As the final GMO content is presented as a relative value, some factors which affect absolute quantification can be partially ignored if they affect both the target and the endogene in the same way. The advantage of using absolute quantification of GMOs (transgene and endogene) is in the elimination of the need for a standard curve generated from certified reference materials. Cost-efficiency is currently still on the qPCR side for simplex assays; however, multiplex assays shift the cost-efficiency towards dPCR.

 

Source:

http://doi.org/10.1007/s00216-018-1010-1

 

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