Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms

Abstract

Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called 'rain'. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis. We developed an Excel based 'experience matrix' that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations. The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.

Keywords: ABI, LifeTechnologies (formerly AppliedBiosystems); Bio, DNA Technology/Biosearch Technologies; Cat. No., catalogue number; DNA, deoxyribonucleic acid; Droplet digital PCR (ddPCR); EC, European Commission; ERM, Certified European Reference Material; EU, European Union; EURL-GMFF, European Reference Laboratory for GM Food and Feed; Experience matrix; Food/feed analysis; GM, genetically modified; GMO, genetically modified organism; Genetically modified organism (GMO); HEX,H, hexachlorfluorescein; L, liter; Lec, lectin gene of soy; MIQE, minimal information for publication of quantitative digital PCR experiments; MRPL, minimum required performance limit; MS, Microsoft; MWG, Eurofins-MWG; MeanSignal, mean fluorescence signal value; PCR, polymerase chain reaction; Quantification; SD, standard deviation (of fluorescence signals); TAMRA,T, tetramethylrhodamin; TIB, TIB Molbiol; Tech, technician; VBA, visual basic for applications; VIC,V, fluorescent dye (LifeTechnologies); cp/cp, (gene) copy per (gene) copy; dPCR, digital PCR; ddPCR, droplet digital PCR; fluorescein, FAM,F; gDNA, genomic DNA; qPCR, (quantitative) real-time PCR.

Graphical abstract

Fig. 1

Droplet recovery: The figure shows the numbers of accepted droplets (ordinate) for experiments on GMO analysis in our laboratory (abscissa; the numbers were given consecutively for all 2800 PCR reactions performed, duplex reactions were counted only once). Green triangles represent droplet populations transferred with a manual 1-channel pipette to the 96-well PCR plate, blue diamonds represent droplet populations transferred with an automatic 8-channel pipette; both were handled by the same technician (Tech1). Red and purple squares represent automatically transferred populations by two other technicians (Tech2, Tech3). (1) and (2) are outliers, while (a) and (b) represent shifts (see Section 3.1).

Fig. 2

Temperature gradients for four GM soy assays – Droplet view: The figure shows the droplet populations for single assays run in duplex: fluorescence amplitude (ordinate) for each droplet (abscissa). The names and percentage of the soy events measured are given on the left. Blue and green dots represent the positive droplets (above the pink horizontal threshold) for transgene and reference gene, respectively. Grey dots represent the negative droplets. A temperature gradient was applied for both the transgene and reference gene assays.

Fig. 3

Discrimination between positive and negative droplets: Measurement of soy event 356043 1% ERM (BF425c). The figure shows the droplet populations for single assays run in duplex: fluorescence amplitude (ordinate) for each droplet (abscissa). False colours represent the droplet concentration (blue for low and red for high concentrations). A temperature gradient from was applied for both the transgene and reference gene assays.

Fig. 4

Detailed effects of threshold setting and annealing temperature on measured GMO content: Results for measurement of soy event 356043 10% ERM (BF425c) for three annealing temperatures (Figure 3). Thresholds for transgene and reference gene are given on the ordinate or abscissa, respectively. GMO percentages deviating by a maximum of 2% from manually pre-defined thresholds (coloured in orange), are marked in green. The underlying target gene concentrations are given for comparison of the border regions.

Fig. 5

Effect of annealing temperature and oligonucleotide concentration: The upper part of the figure shows the droplet populations for single assays run in duplex. The PCR setup was done with two different oligonucleotide concentrations. A temperature gradient was applied for both the transgene and reference gene assays with both oligonucleotide concentrations. The lower part of the figure shows the corresponding condensed information as Pivot charts from the matrix. For symbols refer to Fig. 6.

Fig. 6

Temperature gradients for four GM soy assays – Matrix view: The figure shows Pivot charts from our matrix. Coloured squares represent the approximate background and coloured triangles the approximate signal for the chosen Pivot options. Red, orange, light green and dark green represent none, moderate, good and very good separation, respectively.

Fig. 7

Matrix overview for soy 40-3-2 event and reference gene assays: The figure shows Pivot charts from the matrix for the detection of soy event 40-3-2 under various conditions (single and duplex PCR, normal and high primer and probe concentrations, probes from different suppliers, all at 60 °C annealing/extension temperature). For symbols refer to Fig. 6.