Detection of Soybean GMO Events Using Two Multiplex Droplet Digital PCR Assays

Abstract

Background

Detection methods for GMO events are required because of regulatory compliance requirements. Efficient detection and quantification of GMO events saves time and resources. Multiplex digital PCR (dPCR) allows detection and quantification of more than one GMO event at the same time.

Objective

The study used two tetraplex droplet digital PCR (ddPCR) assays for the detection of 19 soybean GMO events.

Methods

Two multiplex dPCR assays were developed and optimized for the detection of 19 soybean GMO events. The first tetraplex ddPCR assay contained four element-specific targets commonly found in GMO plants (P-35S, T-nos, tE9, and Pat). The second event-specific tetraplex ddPCR assay targeted four soybean GMO events that are not detected with the element-specific tetraplex ddPCR (CV127, DP305423, MON87701, and MON87751).

Results

The element-specific tetraplex ddPCR assay detected all the expected 15 soybean GMO events. The element-specific tetraplex ddPCR assay also detected selected soybean GMO events at the 0.01% level. The event-specific tetraplex ddPCR assay was successfully used to quantify the four soybean GMO events at the 0.1, 1, 2, and 5% levels. The event-specific tetraplex ddPCR assay also detected the four soybean GMO events at the 0.01% level.

Conclusions

The two tetraplex ddPCR assays can be used for the detection of 19 soybean GMO events.

Highlights

An element-specific tetraplex ddPCR assay was used to detect 15 soybean GMO events, and an event-specific tetraplex ddPCR assay was used to detect and quantify four soybean GMO events that are not detected by the element-specific ddPCR assay.

Genetically modified crops have been developed to increase yield, reduce the need for pesticides, enhance resistance to pests and diseases, improve nutrient quality, and increase resistance to abiotic stresses such as drought (1). Detection of genetically modified organisms (GMOs) is necessary to meet regulatory compliance requirements by many countries (2). European Commission Regulation Number 1829/2003 lays down the provision for authorization, supervision, and labeling of genetically modified food and feed. There is no tolerance for the presence of unapproved GMO events in grain shipments to the European Union (EU) and many other countries. In the EU, food products containing authorized GMO material above 0.9% need to be labeled as GMOs (3).

Real-time PCR and digital PCR have been widely used for the detection and quantification of genetically modified organisms (4–6). Element-specific, construct-specific, and event-specific PCR-based methods are generally used for the detection of GMOs (7). For general GMO screening or testing, DNA sequences from regulatory elements (e.g., promoters and terminators) that are commonly found in many GMO constructs are used (8). Event-specific PCR enables accurate identification and quantification of GMOs, as one primer is located within the GM construct, whereas the other is located in the plant’s own DNA. Digital PCR, unlike real-time PCR, does not necessitate a standard curve for the quantitative analysis of GMOs, making it a preferred method. Reliable reference material (seed, ground material, or DNA) is needed for accurate analysis of GMOs using real-time PCR. Digital PCR has also been reported to be less sensitive to inhibitors present in the DNA (9). Droplet digital PCR (ddPCR; emulsion based) and chip-based dPCR (cdPCR, microfluidics) are the commonly used digital PCR platforms (10). The PCR solution is divided into thousands of droplets in the case of ddPCR. Each droplet is amplified by PCR, and a droplet-reading instrument is used to distinguish between positive and negative droplets. Different kinds of ddPCR instruments can be used for GMO detection (11). Many of the ddPCR instruments use a limited number of fluorescent dyes, and optimization is required for the detection of two or more GMO events using multiplex PCR.

Over 40 GMO events have been listed for soybean (12). Many of the GMO events listed are stacked (contain two or more GMO events). Currently, 17 authorized, nine unauthorized, and one expired single soybean GMO events are listed (https://gmo-crl.jrc.ec.europa.eu/jrcgmomatrix/matrices/full).

Detection and quantification of several GMO events at the same time increases efficiency and saves resources (13, 14). Digital PCR has been used for the detection of multiple GMO events (15, 16). Detection and quantification of two, three, and four GMO events at the same time has also been achieved (17).

The objective of the present study is to detect 19 soybean GMO events using two multiplex droplet digital PCR (ddPCR) assays. Four element-specific and four event-specific assays were included for the two multiplex ddPCR assays. The element-specific tetraplex ddPCR assay detects 15 soybean GMO events, and the tetraplex event-specific ddPCR detects four GMO events that are not detected by the element specific ddPCR assay. The two tetraplex ddPCR assays will enable rapid testing of soybean samples for the presence of GMO events.

Experimental

Sources of Reference Materials

The certified reference materials used for soybean ddPCR assays are provided in Table 1. Certified seeds of Colby (non-GMO variety) were obtained from WG Thompson & Sons (Ontario, Canada).

Table 1.

List of 19 soybean GMO events, markers, and reference materials used for multiplex ddPCR detection

	Marker used	Reference materials
GMO event	for detection	Unique code	Supplier
A2704-12	P-35S, Pat	AOCS0707-B15	AOCSa
A5547-127	P-35S, Pat	AOCS0707-C5	AOCS
CV127	Event-specific	AOCS0911-C	AOCS
DAS44406-6	Pat	ERM-BF436bb	SIGMA
DAS68416-4	Pat	ERM-BF432d	SIGMA
DAS81419-2	Pat	ERM-BF437b	SIGMA
DBN-09004–6	tE9	ERM-BF441B	LGC Standards
DP305423	Event-specific	ERM-BR426d	SIGMA
DP356043	P-35S	ERM-BF425d	SIGMA
FG72	T-nos	AOCS0610-A3	AOCS
GMB151	P-35S	ERM-BF443b	SIGMA
MON40-3–2	P35-S, T-nos	ERM-BF410gk	SIGMA
MON87701	Event-specific	AOCS0809-A	AOCS
MON87705	tE9	AOCS0210-A	AOCS
MON87708	tE9	AOCS0311-A	AOCS
MON87751	Event-specific	AOCS0215-A	AOCS
MON87769	tE9	AOCS0809-B2	AOCS
MON89788	tE9	AOCS0906-B	AOCS
SYHTOH2	P35S, T-nos, Pat	AOCS0112-A2	AOCS

a

AOCS = American Oil Chemists’ Society.

b

ERM = European Reference Material.

DNA Extraction

DNA from both GMO and non-GMO sources was extracted using the DNeasy mericon Food kit (Qiagen Life Sciences, LLC, Louisville, KY). DNA dilution and quantification was as described (17). After DNA quantification, solutions of each GMO and non-GMO stock were prepared at 20 ng/μL concentration. A DNA mixture consisting of the different GMO events was prepared for the tetraplex ddPCR assays. For element-specific tetraplex ddPCR, DNA for each of the 15 GMO events was prepared at 1% and tested using the tetraplex ddPCR assay. In addition, the LOD of the tetraplex element-specific ddPCR assay was evaluated using 0.01 and 0.05% DNA mixtures from four soybean GMO events. To prepare DNA mix, four GMO events were added at 5% each (20% in total) to 80% of non-GMO DNA. For PCR set up, 5 μL (100 ng) of the tetraplex DNA mix was used. Dilutions for lower concentrations (e.g., 0.1 and 0.01%) were made from the 5% stock.

Droplet Digital PCR

A Bio-Rad QX200 instrument was used for ddPCR. A Bio-Rad ddPCR Multiplex Supermix was used for the multiplex ddPCR as described (18). The QuantaSoft 1.7.4.0917 and automatic threshold were used for the ddPCR assays. PCR amplification of the generated droplets was carried out using an Eppendorf Master Cycler (Sigma-Aldrich). 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/s was used at the last step to maintain the reaction at 15°C.

The four element-specific targets used were: Cauliflower Mosaic Virus 35S (P-35S, 19), nopaline synthase terminator (T-nos, 19), E9 terminator (tE9, 20), and phosphinothricin N-acetyl transferase (Pat, 20). The 15 soybean GMO events that are detected with the element-specific ddPCR assays are listed in Table 1. The four soybean GMO events, CV127, DP305423, MON87701, and MON87751, do not have any of the four element-specific targets (https://gmo-crl.jrc.ec.europa.eu/jrcgmomatrix/matrices/full). Thus, an event-specific multiplex ddPCR assay was established for the detection of the four soybean GMO events. DNA concentrations of 0.1, 1, 2, and 5% were used for ddPCR to determine the accuracy of the tetraplex event-specific ddPCR assay. The element-specific, event-specific, and the reference gene (Lectin) primer and probe DNA sequences, amplicon sizes, and the optimized concentrations of primers and probes used are provided in Table 2. Optimization of tetraplex ddPCR assays was done by adjusting the primer and probe concentrations. DNA mixtures from the four soybean GMO events (CV127, DP305423, MON87701, and MON87751) were prepared at 0.1, 1, 2, and 5% and used for tetraplex event-specific ddPCR. During optimization of the tetraplex element-specific ddPCR assay, 1% DNA from each of the 15 GMO events was tested. Four replications of the reference Lectin gene assay were run separately on the same plate with the ddPCR assay. For calculation of percentage target molecules, the number of positive target droplets was divided by the number of positive reference gene droplets. The droplets were counted using the droplet reader of the QX200 system. QX Manager Software (version 1.2) was used for analyzing tetraplex ddPCR assays. The QX Manager Software fits the fraction of positive droplets to a Poisson algorithm to determine the starting concentrations of the target DNA molecule. DNA concentrations of 0.01 and 0.05% were used to assess the limit of detection for element-specific and event-specific tetraplex ddPCR assays. For the tetraplex event-specific ddPCR assay, the coefficient of variation and bias were calculated for 0.1, 1, 2, and 5% spiked DNA samples.

Table 2.

DNA sequences for primers/probes and concentrations used for multiplex ddPCR

NAME	DNA sequence (5′-3′)	Amplicon size, bp	Conc., μM
35S FTM	GCC-TCT-GCC-GAC-AGT-GGT	82	0.1
35S RTM	AAG-ACG-TGG-TTG-GAA-CGT-CTT-C		0.1
35S TMP	HEX-CAA-AGA-TGG-ACC-CCC-ACC-CAC-G-BHQ1		0.1
180-F (T-nos)	CAT-GTA-ATG-CAT-GAC-GTT-ATT-TAT-G	84	0.4
180-R (T-nos)	TTG-TTT-TCT-ATC-GCG-TAT-TAA-ATG-T		0.4
TM180 YY (P)	FAM-ATG-GGT-TTT-TAT-GAT-TAG-AGT-CCC-GCA-A-BHQ1		0.22
Pat-F	CGC-GGT-TTG-TGA-TAT-CGT-TAA-C	108	0.4
Pat-R	TCT-TGC-AAC-CTC-TCT-AGA-TCA-TCA-A		0.4
Pat-P	FAM-AGG-ACA-GAG-CCA-CAA-ACA-CCA-CAA-GAG-TG-BHQ1		0.12
tE-9-F	TTT-GTT-GTG-CTT-GTA-ATT-TAC-TGT-GTT	75	0.34
tE-9-R	TTC-TCC-ATC-CAT-TTC-CAT-TTC-A		0.34
tE-9-P	HEX-TTT-ATT-CGG-TTT-TCG-CTA-TC-BHQ1		0.32
CV127- SE-127 F	AAC AGA AGT TTC CGT TGA GCT TTA AGA C	88	0.4
CV127- SE-127 R	CAT TCG TAG CTC GGA TCG TGT AC		0.4
CV127- SE-127 P	FAM-TTT GGG GAA GCT GTC CCA TGC CC–BHQ1		0.06
DP305-f1	CGT GTT CTC TTT TTG GCT AGC	93	0.8
DP305-r5	GTG ACC AAT GAA TAC ATA ACA CAA ACT A		0.5
DP305-p	FAM-TGA CAC AAA TGA TTT TCA TAC AAA AGT CGA GA BHQ1		0.16
MON87701 F	TGG TGA TAT GAA GAT ACA TGC TTA GCA T	89	0.6
MON87701 R	CGT TTC CCG CCT TCA GTT TAA A		0.6
MON87701 P	HEX-TCA GTG TTT GAC ACA CAC ACT AAG CGT GCC–BHQ1-3’		0.16
MON87751 F	CTA AAT TGC TCT TTG GAG TTT ATT TTG TAG	87	0.5
MON87751 R	GGC CTA ACT TTT GGT GTG ATG ATG		0.5
MON87751 P	HEX-TGA CTG GAG ATC TCC AAA GTG AGG GGA AA–BHQ1		0.12
LecF	CCA GCT TCG CCG CTT CCT TC	74	0.4
LecR	GAA GGC AAG CCC ATC TGC AAG CC		0.4
LecP	FAM-CTT CAC CTT CTA TGC CCC TGA CAC-BHQ1		0.2

Results and Discussion

Optimization of the Multiplex ddPCR Assays

For the initial tetraplex element-specific ddPCR assay, recommended primer and probe concentrations were used (19, 20). For each detection assay, FAM and HEX probes were used to confirm amplification and observe cluster location. After the determination of the best event pairing for each probe label, low and high positions of the clusters were assigned on the amplitude plot. Tetraplex ddPCR was run to determine cluster separation for the element-specific and event-specific ddPCR assays. Primer and probe concentrations were adjusted for each of the tetraplex ddPCR assays to obtain clear separation of the 16 clusters. The optimized concentrations of primers and probes used for both tetraplex ddPCR assays are provided in Table 2. Droplet clusters were not clearly separated at the beginning of the tetraplex ddPCR assay (Figure 1A). However, clear separation of clusters was observed after optimization of primer and probe DNA concentrations (Figure 1B).

Figure 1.

Optimization of element-specific tetraplex ddPCR assay. Figure 1A shows distribution of droplets before optimization. Figure 1B shows distribution of droplets for optimized tetraplex ddPCR assay (5% DNA mix). A = P-35S; B = tE9; C = Pat; and D = T-nos.

Tetraplex Event-Specific and Element-Specific ddPCR Assays

The event-specific tetraplex ddPCR assay included the soybean GMO events CV127, DP305423, MON87701, and MON87751. Spiked DNA samples of the four GMO events were analyzed with the tetraplex event-specific ddPCR assay. Expected values were obtained for 1, 2, and 5% spiked DNA samples (Table 3). There was a tendency for a higher bias for 0.1% spiked DNA samples. For low GMO concentrations such as 0.1%, the relative bias tends to be higher (21). The bias for 1, 2, and 5% GMO concentrations was below 25%, with the exception of 1% MON87751. The coefficient of variation for all samples was ≤16% (Table 3). A two-dimensional view of distribution of droplets for 5% GMO samples for the event-specific ddPCR assay is shown in Figure 2. There was clear separation of the negative droplets and the 15 possible positive droplet clusters. Each of the 15 GMO events (listed in Table 1) that contain one or more of the four element-specific DNA sequences (P-35S, T-nos, tE9, and Pat) were tested with the element-specific tetraplex ddPCR assay at 1% level. Amplification was achieved for each of the 15 GMO events, indicating that the tetraplex element-specific ddPCR assay is suitable for the detection of the GMO events. The event-specific tetraplex ddPCR assay was used to detect and quantify four soybean GMO events that were not detected by the element-specific tetraplex ddPCR assay. Thus, the two tetraplex ddPCR assays can detect the 19 soybean GMO events.

Figure 2.

Distribution of droplet clusters for tetraplex event-specific ddPCR assay (5% sample mix). A = MON87751; B = MON87701; C = CV127; and D = DP305423. The dark cluster represents negative droplets.

Table 3.

Quantitative results for tetraplex event-specific ddPCR assay^a

			Coefficient
Event	Concentration	Mean, %	of variation, %	Bias, %
CV127	0.1%	0.14 ± 0.02	10.5	40
CV127	1%	1.21 ± 0.08	6.7	21
CV127	2%	2.37 ± 0.10	4.2	18.5
CV127	5%	5.37 ± 0.30	5.6	7.5
DP305423	0.1%	0.14 ± 0.02	15.6	40
DP305423	1%	1.15 ± 0.07	6.5	14.8
DP305423	2%	2.2 ± 0.10	4.7	10
DP305423	5%	4.92 ± 0.22	4.5	1.6
MON87751	0.1%	0.16 ± 0.02	12.0	61.7
MON87751	1%	1.29 ± 0.07	5.2	28.8
MON87751	2%	2.37 ± 0.10	4.1	18.5
MON87751	5%	5.34 ± 0.37	6.9	6.8
MON87701	0.1%	0.15 ± 0.02	13.3	50
MON87701	1%	1.22 ± 0.10	8.0	21.7
MON87701	2%	2.22 ± 0.11	4.9	11
MON87701	5%	5.18 ± 0.21	4.1	3.5

a

Each value is an average of six replications ± standard deviation.

Evaluation of Limits of Detection

Low GMO concentrations were tested to determine the limit of detection of the two multiplex ddPCR assays. For the element-specific tetraplex ddPCR assay, 0.01 and 0.05% spiked DNA samples were prepared and PCR carried out with 20 replications on two different dates (Table 4). For tetraplex element-specific ddPCR that included DAS81419 (Pat), FG72 (T-nos), DP356043 (P-35S), and MON87705 (tE9), an average of 5 to 12 droplets were generated for 0.01% spiked DNA samples. For 0.05% spiked DNA samples, an average of 17 to 43 droplets were generated (Table 4). For the event-specific tetraplex ddPCR assay (CV127, DP305423, MON87701, and MON87751 soybean GMO events), an average of four to six droplets were generated for 0.01% spiked DNA samples. For the 0.05% spiked DNA sample, an average of 17 to 22 droplets were generated (Table 4). The results of experiments conducted on two different dates were similar, demonstrating consistency. According to Niu et al. (22), a threshold of five droplets was used to determine a positive replicate. Based on the number of droplets generated, the limit of detection for both element-specific and event-specific tetraplex ddPCR assays is 0.01%. The number of droplets generated for the 0.05% samples was much higher than for the 0.01% samples, but still fewer than the theoretical fivefold difference. The LOD is for a qualitative test, and the fivefold difference will not have an impact on the results. There was some variability in the total number of droplets generated, which may have contributed to the discrepancy.

Table 4.

Detection of low concentrations of spiked GMO DNA with element and event-specific tetraplex ddPCR assays

		Average number of positive droplets
ddPCR Assay	Target	0.01% A	0.01% B	0.05% A	0.05% B
Tetraplex	P35S	7.1 ± 2.8	7 ± 2.2	24.9 ± 6.4	25 ± 6.4
element-specifica	Pat	12.1 ± 5.7	7 ± 3.0	23.7 ± 9.5	18 ± 5.6
	tE9	4.7 ± 2.5	5 ± 2.8	17.0 ± 7.3	17 ± 7.3
	T-nos	11.8 ± 3.7	12 ± 3.7	43.3 ± 9.8	43 ± 9.7
Tetraplex	CV127	6.1 ± 3.4	5.6 ± 2.4	20.7 ± 5.5	21.7 ± 4.9
event-specific	DP305423	4.0 ± 2.4	3.9 ± 2.2	17.8 ± 4.1	20.2 ± 5.3
	MON87701	5.5 ± 2.6	5.6 ± 1.8	17.3 ± 5.3	21.9 ± 6.0
	MON87751	5.0 ± 2.7	5.8 ± 2.1	19.8 ± 3.4	22.4 ± 5.0

a

DNA from soybean GMO events DP356043, DAS81419, MON87705, and FG72 were used for P35S, Pat, tE9, and T-nos, respectively. A and B indicate experiments carried out on different dates. Each value is the average of 20 replications ± standard deviation.

Applications of the Two Multiplex ddPCR Assays

Faster and more efficient methods are needed to detect the increasing number of soybean GMO events. The four screening elements used for the detection (P-35S, T-nos, Pat, and tE9) are available in many soybean and other GMO plants. The tetraplex element-specific ddPCR assay detected the 15 soybean GMO events listed in Table 1. In addition, the element-specific ddPCR assay can potentially detect the unauthorized soybean GMO events, DBN8002, HB4, MON87712, and Shzd32-01. The P-35S element-specific primer set has been predicted to amplify the four unauthorized soybean GMO events (https://gmo-crl.jrc.ec.europa.eu/jrcgmomatrix/matrices/full). Reference materials were not available to test the four unauthorized soybean GMO events at the time the experiment was conducted. Multiplex ddPCR using the screening elements 35S, NOS, NPTII, and Pat has been used for high-throughput screening of maize GMO events (22). Multiplex ddPCR targeting 35S and NOS terminator sequences was also developed for maize GMO events (16). The multiplex element-specific ddPCR assay described in this study will be useful for testing soybean samples for the presence of GMOs. The tetraplex element-specific ddPCR assay can also be used for the detection of multiple GMO events in other crops such as canola. The event-specific tetraplex ddPCR assay detected the four soybean GMO events at different concentrations. The two multiplex ddPCR assays can be used for the detection of soybean GMO events in grain shipments.

Conclusions

Two tetraplex ddPCR assays (element-specific and event-specific) were used for the detection of 19 soybean GMO events. The element-specific tetraplex ddPCR assay included four commonly used targets for GMO detection (P-35S, T-nos, Pat, and tE9). The element-specific tetraplex ddPCR assay was successfully used for the detection of 15 soybean GMO events. The event-specific tetraplex ddPCR assay targeted four soybean GMO events (CV127, DP305423, MON87701, and MON87751) that are not detected by the element-specific tetraplex ddPCR assay. Different concentrations of spiked GMO DNA samples were successfully quantified with the event-specific ddPCR assay. The limit of detection for both tetraplex ddPCR assays was 0.01%.

CRediT Author Statement

Tigst Demeke: conceptualization, guidance, and preparation of manuscript; Monika Eng: carried out and optimized the PCR experiments, prepared figures and tables, and reviewed the manuscript.

Disclaimer

This document is provided for scientific purposes only. Any reference to a brand or trademark herein is for informational purposes only and is not intended for a commercial purpose or to dilute the rights of the respective owner(s) of the brand(s) or trademark(s).

Conflict of Interest

The authors declare that there is no conflict of interest.

Funding

The authors declare no specific funding for this project.