Food Control 115 (2020) 107291

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Assessment of genetically engineered events in heat-treated and non-treated T

samples using droplet digital PCR and real-time quantitative PCR
Tigst Demekea,∗, Brian Beecherb, Monika Enga
a
Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada
b
USDA-AMS-FGIS, Technology and Science Division, Kansas City, MO, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Regulations and labeling laws established by many countries for genetically engineered (GE) events necessitate
Genetically engineered the development of effective testing methods. Both real-time quantitative PCR (RT-qPCR) and digital PCR
Digital PCR (dPCR) have been used for the detection and quantification of GE events. The advantage of digital PCR is that
Real-time quantitative PCR there is no need to use either a standard curve or reference materials to perform the analysis. In the case of RT-
Heat-treated samples
qPCR, incorrect results may be obtained if the standard curve does not reflect an amplification efficiency be-
Detection
tween 90 and 110%. DNA of high purity is required for obtaining accurate results from both PCR platforms. Food
processing results in the degradation of DNA, which may affect PCR quantification of GE traits. In this study, the
simulated effect of processed vs. non-processed samples on the testing of GE traits was evaluated using both
droplet digital PCR (ddPCR) and RT-qPCR. Ground canola and soybean samples were heat-treated in boiling
water for 15, 30 and 60 min in order to cause DNA degradation to determine the impact on quantification using
both ddPCR and RT-qPCR. The measured ddPCR concentrations were similar to the gravimetric fortification by
weight for three of the four GE events tested for both treated and non-treated samples. For one event, the
concentrations obtained for treated samples were higher by roughly a factor of two than the expected 0.1% and
1% by weight target values. RT-qPCR assays gave similar results to those obtained using ddPCR.

1. Introduction is no need to use standard curve and reference materials. Conversely,

Identification and quantification of genetically engineered (GE)
events is routinely utilized due to established regulations and labeling
requirements of many countries. Food processing results in DNA de-
gradation, which can affect PCR results. Varying effects of food pro-
cessing on PCR results have been reported. Degraded DNA templates
can cause the amount of GE material in a given sample to be under-
estimated (Gryson, 2010). The impact of heat processing on the de-
tection of GE traits using RT-qPCR has been reported (Vijayakumar,
Martin, Gowda, & Prakash, 2009). The extent of DNA degradation is a
critical factor in determining whether a specific sequence can be am-
plified. Conversely, it has been reported that processing does not affect
RT-qPCR of transgenic content in foods (Ballari & Martin, 2013;
Bergerová, Hrnčírová, Stankovská, Lopašovská, & Siekel, 2010;
Fernandes et al., 2016). RT-qPCR results were not affected despite DNA
degradation due to thermal processing at 100 °C, as both transgene and
endogene amplifications were equally affected (Ballari & Martin, 2013).
Droplet digital PCR (ddPCR) has gained popularity for absolute
quantification of GE events. The main advantage of ddPCR is that there
the success of RT-qPCR is dependent upon the quality of reference
material used to generate a standard curve. Optimization of ddPCR,
including comparison with a RT-qPCR method is important in order to
have proper identification and quantification of GE events. Although
information on the impact of degraded DNA on RT-qPCR results is
available, the effect of degraded DNA on ddPCR results has not yet been
investigated. The objective of the study was to determine the effect of
DNA extracted from heat-treated vs. non-treated samples on GE trait
quantification of four events using RT-qPCR and ddPCR.
2. Materials and methods
2.1. Seed sources and heat-treatment
GE and non-GE seed samples were ground with a Retsch model ZM
100 Centrifugal Grinding Mill (Fischer Scientific, Ottawa, Canada)
using a 1 mm sieve. For canola, spiked flour samples were prepared
using the varieties Legend (non-GE variety), Conquest (GT73 event,
breeder seed) and Innovator (HCN92 GE event, breeder seed). The

∗
Corresponding author.
E-mail addresses: tigst.demeke@grainscanada.gc.ca (T. Demeke), Brian.S.Beecher@usda.gov (B. Beecher), monika.eng@grainscanada.gc.ca (M. Eng).

https://doi.org/10.1016/j.foodcont.2020.107291
Received 10 December 2019; Received in revised form 28 March 2020; Accepted 31 March 2020
Available online 01 April 2020
0956-7135/ Crown Copyright © 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T. Demeke, et al. Food Control 115 (2020) 107291

seeds were obtained from the Oilseeds Program of the Canadian Grain
Commission. For soybean, spiked flour samples were prepared using
seeds of the varieties, Colby (non-GE variety), Renwick (GTS-40-3-2 GE
event) and PS 2295 LL (A2704 GE event). Gravimetric fortification was
done at 0.1% and 1% levels. Certified seeds of Colby and Renwick were
obtained from WG Thompson & Sons (Ontario, Canada), while certified
seeds of PS 2295 LL were obtained from Bayer Crop Science.
Heat treatment of the spiked ground samples was carried out ac-
cording to Moreano, Busch, and Engel (2005). Briefly, 16-mL of dis-
tilled deionized water was added to 8 g of ground sample and mixed.
The sample slurry was kept in boiling water for 15, 30 and 60 min time-
points. After boiling, the samples were dried at 45 °C and kept at 4 °C
until DNA extraction was performed. A non-treated sample was used as
a control for each spike level.
2.2. DNA extraction and quantification
DNA was extracted from all samples using the DNeasy mericon Food
kit (Qiagen Sciences, LLC, Louisville, KY). DNA was extracted from 12
heat-treated and non-treated ground samples (0.2 g each). The ground
samples consisted of 0.1% and 1% spiked HCN92 canola, GT73 canola,
GTS-40-3-2 soybean, A2704 soybean and non-GE canola and soybean.
DNA quality was verified by agarose gel-electrophoresis. The 12 DNA
samples extracted for each treatment were combined in order to have
sufficient DNA for ddPCR and RT-qPCR. The SpectraMax M5 spectro-
photometer (MDS Analytical Technologies, Toronto, Canada) was used
to quantify DNA. A portion of the DNA was sent to USDA-AMS-FGIS for
RT-qPCR analysis. A comparison of DNA quantification was conducted
for two GE events using the SpectraMax M5, and NanoDrop One
(ThermoFisher Scientific, Mississauga, ON) ultraviolet spectro-
photometers, and the Pico Green fluorescence assay (Molecular Probes,
Eugene, OR).
2.3. Droplet digital PCR
Digital PCR was carried out at Canadian Grain Commission using
the QX200 ddPCR system (Bio-Rad, Pleasanton, CA). The primer and
probe DNA sequences for the target and reference genes as well as the
concentrations used for ddPCR and RT-qPCR are listed in Table 1.
Duplex ddPCR (mixing of target and reference primers and probes in
the same reaction well) was used to generate target and reference
droplets at the same time. 12.5 μL Bio-Rad ddPCR Supermix for Probes
(no dUTP) and 100 ng template DNA was used for the ddPCR assays.
The DG-32 cartridge for automated droplet generator (Cat. No.
186–4108) was used to generate droplets of 22 μL volume. The gen-
erated droplets were transferred to semi-skirted 96 well Eppendorf
plates (Cat. No. 12001925) and sealed with a pierceable foil heat seal
(Cat. No. 1814040). PCR amplification of the generated droplets was
carried out using a MJ Thermal Cycler (PTC 200). The thermal cycling
conditions used were: an initial denaturation step of 95 °C for 10 min,
followed by 50 cycles of 95 °C for 15 s, 60 °C for 1 min, and a final step
of 98 °C for 10 min. A ramp rate of 0.6 °C/s was used between the
cycling steps and a ramp rate of 0.3 °C/sec was used at the last step to
maintain the reaction at 15 °C. The droplets were counted using the
droplet reader on the QX200 system. QuantaSoft version 1.7.4.0917
and automatic threshold were used for ddPCR data analysis.
2.4. Real-time quantitative PCR
Real-time PCR was carried out using the ABI 7500 PCR instrument
at the Canadian Grain Commission laboratory, and using a QuantStudio
7 instrument at the USDA-AMS-FGIS laboratory (Thermo Fisher
Scientific). ABI 7500 software version 2.3 and QuantStudio™ Real-time
PCR Software version 1.3 were used for RT-qPCR data analysis. The
real-time PCR methods for the four GE events were validated by
European Reference Laboratory for Genetically Modified Food and Feed
(source of information is provided in Table 1 and in the list of refer-
ences). The limit of quantification (LOQ) values for HCN92, GT73, GTS-
40-3-2 and A2704-12 were reported as ≤0.09%, 0.085%, 0.09% and
0.045%, respectively. The reported limit of detection (LOD) values for
each validated method were lower than the LOQ values. The 25 μL RT-
qPCR contained 100 ng DNA template (5 μL of 20 ng/μL DNA solution),
primers and probes (concentrations provided in Table 1 ), and 1X
(12.5 μL) TaqMan Universal Master Mix II with UNG (Applied Biosys-
tems). The thermal profile used for the real-time PCR had an initial hold
for 2 min at 50 °C, 10 min at 95 °C, and then 40 cycles of 15 s at 95 °C

Table 1
List of primers/probes used for real-time quantitative PCR and digital PCR.
Event/reference gene Names of primers/probes DNA sequences (5’ – 3′) Amplicon size (bp) Primer/probe concentrations (μM)

ddPCR RT-qPCR

GT73 canola RT73 primer 1 F-CCA TAT TGA CCA TCA TAC TCA TTG CT 108 0.4 0.15
GT73 canola RT73 primer 2 R-GCT TAT ACG AAG GCA AGA AAA GGA 0.4 0.15
GT73 canola RT73 probe P-FAM-TTC CCG GAC ATG AAG ATC ATC CTC CTT-BHQ1 0.2 0.05
HCN92 canola MDB685-F F-GTT GCG GTT CTG TCA GTT CC 95 0.40 0.40
HCN92 canola KVM180-R R-CGA CCG GCG CTG ATA TAT GA 0.40 0.40
HCN92 canola TM029 probe FAM-TCC CGC GTC ATC GGC GG-BHQ1 0.20 0.20
FatA(A) 09-0-2824 F-ACA GAT GAA GTT CGG GAC GAG TAC 84 0.3 0.3
FatA(A) 09-0-2825 R-CAG GTT GAG ATC CAC ATG CTT AAA TAT 0.9 0.9
FatA(A) 09-QP-87 P-HEX- AAG AAG AAT CAT CAT GCT TC-BHQ1 0.15 –
FatA(A) 09-QP-87 FAM- AAG AAG AAT CAT CAT GCT TC-MGBNFQ – 0.15
GTS-40-3-2 soy 40-3-2 AF TTC ATT CAA AAT AAG ATC ATA CAT ACA GGT T 84 0.4 0.15
GTS-40-3-2 soy 40-3-2 AR GGC ATT TGT AGG AGC CAC CTT 0.4 0.15
GTS-40-3-2 soy 40-3-2 AP FAM-CCT TTT CCA TTT GGG-MGBNFQ 0.2 0.05
A2704-12 soy KVM175 GCA AAA AAG CGG TTA GCT CCT 64 0.4 0.4
A2704-12 soy SMO001 ATT CAG GCT GCG CAA CTG TT 0.4 0.4
A2704-12 soy TM031 FAM-CGG TCC TCC GAT CGC CCT TCC-TAMRA – 0.2
A2704-12 soy TM031 FAM-CGG TCC TCC GAT CGC CCT TCC-BHQ1 0.2 –
Le1 for 40-3-2 lec-F CCA GCT TCG CCG CTT CCT TC 74 0.4 0.15
Le1for 40-3-2 lec-R GAA GGC AAG CCC ATC TGC AAG CC 0.4 0.15
Le1for 40-3-2 lec-P VIC-CTT CAC CTT CTA TGC CCC TGA CAC-BHQ1 – 0.05
Le1for 40-3-2 lec-P VIC-CTT CAC CTT CTA TGC CCC TGA CAC-BHQ1 0.2

Note: In both ddPCR and RT-PCR, the FatA(A) and Le1 reference gene sequences were used for canola, and soybean, respectively. Sources for primers and probes: GT73 =
EURL GMFF (2007a) – line RT73; HCN92 = EURL GMFF (2011)–line TOPAS 19/2; FatA(A) = Henderson, Harmon, & Zhong, 2016; GTS-40-3-2 = EURL GMFF (2009)
– line 40-3-2; A2704-12 & Le1 = EURL GMFF (2007b) – line A2704-12 (see references for details).

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T. Demeke, et al. Food Control 115 (2020) 107291

Fig. 1. Example of DNA extracted from non-treated and heat-treated samples
(1.5% agarose gel). M = Low DNA mass ladder (2 μL of 117.5 ng/μL);
1–4 = 1% GT73 canola DNA; 5–8 = 1% HCN92 canola DNA. 1 & 5 = DNA
from non-treated samples; 2 & 6 = DNA from 15 min heat-treated samples; 3 &
7 = DNA from 30 min heat-treated samples; 4 & 8 = DNA from 60 min heat-
treated samples; and 9 = Lambda DNA (100 ng). 300 ng of genomic DNA was
loaded on the gel.
average DNA yield extracted from non-treated samples was higher than
that of heat-treated samples. Similarly, it has been reported that the
amount of DNA obtained in autoclaved samples was much lower than
that of non-autoclaved samples (Vijayakumar et al., 2009). The
Abs260/280 ratios for the extracted DNA were between 1.7 and 2.4
(Table 2). The Abs260/280 ratio for pure DNA is ~1.8 (Matlock, 2015).
The concentration of DNA was also measured for two events using
three methods: SpectraMax M5 spectrophotometer (UV-based),
NanoDrop One (UV-based), and the PicoGreen assay. A higher con-
centration of DNA was observed using NanoDrop One compared with
the SpectraMax M5 measurement (Table 3). For all heat-treated sam-
ples, the concentration of DNA was substantially underestimated with
the PicoGreen assay method. The PicoGreen assay is a fluorescent-based
double stranded DNA quantification method. Most of the degraded DNA
is likely single stranded, which cannot be measured with the PicoGreen
assay method. Underestimation of DNA concentration using the Pico-
Green method has been reported by other investigators (Sedlackova
et al., 2013; Shokere et al., 2009), consistent with our results.
3.2. ddPCR and RT-qPCR results for DNA extracted from heat-treated and
non-treated samples

and 1 min annealing at 60 °C. Both labs used the same RT-qPCR con-
ditions.
3. Results and discussion
3.1. Quantification of DNA
One of the challenges of working with degraded DNA is accurate
quantification. Fluorometric and ultraviolet DNA measurements may
not provide the same results (Shokere, Holden, & Jenkins, 2009;
Sedlackova, Pepiska, Celec, Szemes, & Minarik, 2013). Both fluoro-
metric and qPCR-based DNA measurements were significantly influ-
enced by DNA fragmentation. Conversely, the accuracy of ultraviolet
spectrophotometric measurements were not influenced by the level of
DNA fragmentation (Sedlackova et al., 2013). Ultraviolet measurement
(SpectraMax M5 instrument) was chosen to quantify the amount of
DNA used for the PCR analyses in this study. Agarose gel electrophor-
esis demonstrated progressive increases in DNA degradation as the total
time of heat-treatment increased (Fig. 1). In addition, heat-treated
samples contained fractions of DNA larger than 200 bp, which can be
amplified with PCR (Fig. 1). All samples contained a sufficient quantity
of DNA to perform PCR analysis (Table 2). For all four GE events, the
ddPCR results for DNA extracted from non-treated and heat-treated
samples are shown in Table 4. For three of the four GE events (GT73
canola, GTS-40-3-2 soybean and A2704 soybean), close to the 0.1% and
1% expected results were obtained for both heat-treated and non-
treated samples. For the 15 and 30 min heat treatments, the number of
target and reference droplets were higher than the non-treated samples
for both GE soybean events (Table 5). The 60 min heat treatment re-
sulted in fewer numbers of droplets for both target and reference genes
compared with 15 and 30 min treatments, indicating the negative effect
of DNA template degradation on PCR amplification (Table 5). An ex-
ample of an amplitude plot of ddPCR results is provided in Fig. 2. In
general, a greater number of positive targets appeared in dual droplets
(contain both target and reference droplets) versus single droplets.
For the HCN92 canola event, ddPCR and RT-qPCR results from the
non-treated samples provided results close to the 0.1% and 1% target
concentrations (Table 4). However, the GE percentage values obtained
for heat-treated samples were higher than the expected concentrations
for both assays. The 15 and 30 min heat-treated samples had higher
number of target droplets than non-treated samples, but the number of
reference droplets were similar in both heat-treated and non-treated
samples, resulting in overestimation of the percentage GE values
(Tables 4 and 5). The number of target and reference droplets was

Table 2
Average DNA yield and A260/A280 ratios obtained for extracted DNA using SpectraMax M5 instrument.
Average DNA yield (ng/μL) Average A260/A280

Event Treatment 0.1% 1% 0.1% 1%

HCN92 canola NT 132.6 ± 4.2 146.1 ± 0.7 1.8 ± 0.0 1.9 ± 0.0
15 min 73.9 ± 12.3 57.3 ± 4.7 1.8 ± 0.3 2.3 ± 0.1
30 min 54.8 ± 2.0 49.2 ± 1.5 2.1 ± 0.2 2.3 ± 0.2
60 min 67.1 ± 18.7 48.0 ± 2.4 1.7 ± 0.2 2.4 ± 0.1
GT73 canola NT 130.2 ± 1.9 159.1 ± 8.0 1.9 ± 0.1 1.8 ± 0.0
15 min 55.4 ± 5.0 77.4 ± 16.2 2.2 ± 0.2 1.9 ± 0.3
30 min 73.7 ± 3.1 76.2 ± 12.2 2.1 ± 0.0 1.8 ± 0.1
60 min 52.2 ± 3.5 68.6 ± 5.9 1.8 ± 0.5 1.7 ± 0.1
GTS40-3-2 soybean NT 315.7 ± 5.0 240.3 ± 11.7 1.9 ± 0.0 1.9 ± 0.0
15 min 88.9 ± 3.5 120.1 ± 15.8 2.0 ± 0.2 1.8 ± 0.1
30 min 90.5 ± 2.2 90.6 ± 2.4 2.0 ± 0.1 1.9 ± 0.0
60 min 87.5 ± 6.4 84.7 ± 4.5 1.8 ± 0.1 1.8 ± 0.1
A2704 soybean NT 376.8 ± 2.8 407 ± 6.5 2.0 ± 0.0 1.9 ± 0.0
15 min 66.7 ± 3.6 107.2 ± 3.1 1.9 ± 0.3 1.8 ± 0.1
30 min 68 ± 6.4 99.9 ± 3.5 2.1 ± 0.2 1.8 ± 0.0
60 min 76.8 ± 4.4 84.0 ± 5.4 2.0 ± 0.1 1.8 ± 0.1

NT = Non-treated. Average of three replicates plus minus standard deviation.

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T. Demeke, et al. Food Control 115 (2020) 107291

Table 3
Comparison of DNA yield for three DNA quantification methods.
DNA yield (ng/uL)

Event & concentration Treatment SpectraMax M5 NanoDrop PicoGreen

GT73 canola NT 159.13 ± 8.0 210.03 ± 2.24 147.44 ± 2.02

1% 15 min 77.40 ± 16.18 94.37 ± 2.99 19.2 ± 0.09
30 min 76.23 ± 12.2 88.63 ± 0.68 16.44 ± 0.14
60 min 68.60 ± 5.95 81.87 ± 0.81 14.93 ± 0.29
A2704 soy NT 407.00 ± 6.5 560.67 ± 0.21 166.73 ± 2.27
1% 15 min 107.17 ± 3.12 163.5 ± 3.60 29.30 ± 0.14
30 min 99.93 ± 3.5 135.27 ± 3.75 23.88 ± 0.07
60 min 83.97 ± 5.4 107.30 ± 1.21 19.05 ± 0.24

NT = Non-treated. The SpectraMax M5 measurement was used to determine DNA concentrations for both digital and real-time PCR analyses.

Table 4
Droplet digital PCR and RT-qPCR results obtained for DNA extracted from non-treated and heat-treated samples.
0.1% spiked samples 1% spiked samples

Event Heat Treatment ddPCR RT-qPCR1 RT-qPCR2 ddPCR RT-qPCR1 RT-qPCR2

HCN92 canola NT 0.09 ± 0.02 0.08 ± 0.02 0.07 ± 0.01 0.89 ± 0.04 0.59 ± 0.09 0.58 ± 0.02
15 min 0.22 ± 0.02 0.15 ± 0.04 0.13 ± 0.01 2.20 ± 0.07 1.65 ± 0.36 1.49 ± 0.06
30 min 0.32 ± 0.04 0.27 ± 0.05 0.23 ± 0.03 1.85 ± 0.05 1.49 ± 0.34 1.26 ± 0.05
60 min 0.19 ± 0.05 0.18 ± 0.05 0.13 ± 0.02 2.20 ± 0.08 1.82 ± 0.36 1.43 ± 0.08
GT73 canola NT 0.08 ± 0.01 0.06 ± 0.01 0.06 ± 0.02 0.77 ± 0.05 0.65 ± 0.05 0.66 ± 0.04
15 min 0.07 ± 0.01 0.07 ± 0.03 0.06 ± 0.01 0.81 ± 0.06 0.60 ± 0.21 0.75 ± 0.14
30 min 0.07 ± 0.01 0.07 ± 0.02 0.06 ± 0.02 0.82 ± 0.04 0.77 ± 0.18 0.71 ± 0.04
60 min 0.11 ± 0.01 0.09 ± 0.02 0.08 ± 0.02 1.04 ± 0.06 0.79 ± 0.10 0.93 ± 0.07
GTS-40-3-2 soybean NT 0.09 ± 0.01 0.08 ± 0.03 0.08 ± 0.02 1.04 ± 0.07 0.96 ± 0.13 0.89 ± 0.06
15 min 0.08 ± 0.02 0.07 ± 0.02 0.06 ± 0.01 0.77 ± 0.03 0.97 ± 0.17 0.81 ± 0.04
30 min 0.06 ± 0.01 0.07 ± 0.02 0.05 ± 0.01 0.78 ± 0.05 0.96 ± 0.12 0.85 ± 0.06
60 min 0.06 ± 0.02 0.06 ± 0.02 0.06 ± 0.01 0.67 ± 0.03 0.58 ± 0.20 0.61 ± 0.04
A2704 soybean NT 0.10 ± 0.04 0.14 ± 0.04 0.12 ± 0.03 0.90 ± 0.08 1.17 ± 0.30 1.24 ± 0.13
15 min 0.07 ± 0.01 0.03 ± 0.02 0.03 ± 0.01 0.63 ± 0.04 1.04 ± 0.22 0.39 ± 0.07
30 min 0.07 ± 0.01 0.09 ± 0.04 0.06 ± 0.02 0.94 ± 0.02 0.79 ± 0.28 0.65 ± 0.10
60 min 0.08 ± 0.01 0.06 ± 0.03 0.07 ± 0.01 0.98 ± 0.07 1.03 ± 0.19 1.25 ± 0.10

NT = Non-treated. The data is average of six repeats (two different days, three replicates each) plus minus standard deviation. RT-qPCR1 = Research work carried
out at Canadian Grain Commission, Winnipeg, MB. RT-qPCR2 = Research work carried out at USDA-AMS-FGIS (Kansas City, Missouri).

Table 5
Number of target and reference droplets generated for droplet digital PCR.
0.1% spiked samples Number of droplets 1% spiked samples Number of droplets

Event Heat Treatment Target Reference Target Reference

HCN92 canola NT 35 ± 9 16539 ± 642 312 ± 52 16498 ± 1358

15 min 62 ± 7 15120 ± 1035 916 ± 52 17517 ± 1211
30 min 92 ± 11 15261 ± 803 681 ± 29 16265 ± 754
60 min 23 ± 8 8738 ± 1537 381 ± 122 12409 ± 791
GT73 canola NT 29 ± 4 16260 ± 561 269 ± 29 15865 ± 1084
15 min 25 ± 5 15462 ± 1090 209 ± 31 13764 ± 1731
30 min 20 ± 3 15094 ± 654 201 ± 10 13873 ± 545
60 min 19 ± 8 10878 ± 340 131 ± 5 9184 ± 449
GTS-40-3-2 soybean NT 17 ± 3 11889 ± 758 195 ± 10 11471 ± 434
15 min 60 ± 11 16094 ± 1539 461 ± 75 16022 ± 2148
30 min 35 ± 5 15646 ± 1761 442 ± 31 16444 ± 1100
60 min 21 ± 3 14435 ± 1314 239 ± 21 14721 ± 878
A2704 soybean NT 13 ± 5 9027 ± 1895 117 ± 18 9017 ± 838
15 min 60 ± 10 17073 ± 772 407 ± 40 17149 ± 1109
30 min 44 ± 9 16251 ± 763 449 ± 46 16378 ± 542
60 min 30 ± 5 14666 ± 671 263 ± 27 13817 ± 669

NT = Non-treated. The data is average of six repeats (two different days, three replicates each) plus minus standard deviation.

relatively low for the 60 min heated-treated HCN92 samples (Table 5).
For the HCN92 samples, 200 ng of DNA was used instead of 100 ng
DNA for ddPCR in order to determine if DNA quantity was a factor. The
number of target droplets was increased by the use of 200 ng DNA. The
number of reference droplets was also increased, but the percentage GE
values obtained were still overestimated (data not shown). The ex-
pected PCR amplicon size for HCN92 target is 95 bp (Table 1), which is
similar to the amplicon sizes of the other three GE events (Table 1).
Distortion of GE concentration values in heat-treated samples has been
reported (Moreano et al., 2005). Particle size difference was reported to
be the main cause for the distortion in the GE values obtained. Similar
particle sizes were used for GE and non-GE HCN92 samples in this
study. We are not sure why higher than expected GE values were ob-
tained for treated samples. It is important to note that the effect of DNA

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T. Demeke, et al. Food Control 115 (2020) 107291

Fig. 2. Amplitude plot for 1% A2704 soybean. A = Positive droplets for target gene; B = Positive droplets for both target and reference genes; C = Negative
droplets; D = Positive droplets for reference gene. The total number of target droplets for no treatment, 15, 30 and 60 min treatments was 148, 391, 488 and 294,
respectively.

degradation because of heat-treatment may not be the same for all GE
events. Careful assessment is required for each event used and treat-
ment applied.
The RT-qPCR results for the two laboratories were similar to ddPCR
results (Table 4) with minor exceptions. One of the laboratories had low
concentration value for 15 min heat-treated 1% A2704 sample. The
reason for the anomalous result is not clear to us. However, the ddPCR
results were more consistent and close to the expected results compared
to the RT-qPCR results for the 15 min heat-treated 0.1% and 1% A2704
soybean samples.
Some reports have indicated that DNA degradation has a minimal
effect on the quantification of GE events using RT-qPCR (e.g.,
Fernandes et al., 2016), while other studies (e.g., Vijayakumar et al.,
2009) have indicated a negative impact of DNA degradation on the
determination of GE content using RT-qPCR. Vijayakumar et al. (2009)
reported that high temperature and/or pressure used during food

5
T. Demeke, et al.
processing significantly reduced the level of detectable DNA. The copy
number of the transgene decreased when there was high physical da-
mage and fragmentation. For accurate GE content determination of
processed foods, the use of primers that produce short and equal length
of target and reference amplicons has been recommended (Fernandes
et al., 2016; Yoshimura et al., 2005). Bergerová et al. (2010) reported
that heat-processing of maize and soybean samples did not affect real-
time PCR quantification results. DNA fragments ranging from 100 to
200 bp were amplified in all samples. Most of our results are consistent
with previous studies. However, the results may vary for different GE
events as well as on the extent of shearing of the DNA. For highly de-
graded DNA, PCR amplification can be affected, especially if most of the
DNA is below the size of the amplicon to be amplified.
3.3. Summary
DNA quantity and purity is essential for accurate and reproducible
RT-qPCR and ddPCR amplification of DNA from grain samples. Spiked
ground samples of canola and soybean (two GE events for each) were
heat-treated for 15, 30 and 60 min each to investigate the impact of
DNA degradation on ddPCR as well as RT-qPCR. For ddPCR and RT-
qPCR assays, heat-treatment of the samples did not affect the final ex-
pected results for three GE events. For HCN92 canola event, the amount
of GE material was overestimated by a factor of two for the heat-treated
samples. Overall, ddPCR results were more consistent than that of RT-
qPCR results.
CRediT authorship contribution statement
Tigst Demeke: Conceptualization, Writing - original draft, Writing -
review & editing, Supervision. Brian Beecher: Conceptualization,
Writing - review & editing. Monika Eng: Methodology, Formal ana-
lysis.
Declaration of competing interest
The authors have no competing interests to declare.
Acknowledgements
Oilseeds Program of the Canadian Grain Commission is acknowl-
edged for supply of seeds. Drs. Tandace A. Bell and Timothy Norden
(USDA-AMS-FGIS, Kansas City) are acknowledged for reviewing the
Food Control 115 (2020) 107291
paper and providing useful suggestions.
References
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