Skip to main content
NCBI home page
Food Science and Biotechnology logoLink to Food Science and Biotechnology
. 2021 Mar 13;30(4):565–570. doi: 10.1007/s10068-021-00882-3

Detection of GM Canola MS11, DP-073496-4, and MON88302 events using multiplex PCR coupled with capillary electrophoresis

Do-Geun Lee 1, Ji-Eun Park 1, Mi-Ju Kim 1, Hyun-Joong Kim 2, Hae-Yeong Kim 1,✉
PMCID: PMC8050189  PMID: 33936848

Abstract

As of 2020, 11 GM canola events have been authorized as food for humans in Korea. However, there are no simultaneous multiplex detection methods for 3 GM canola events (DP-073496-4, MON88302, and MS11). Thus, we established the multiplex polymerase chain reaction (PCR) method coupled with capillary electrophoresis to detect 3 GM canola events. To verify the specificity of event-specific primers, various GM crops of 3 GM soybean events, 6 GM maize events, 2 GM cotton events and 11 GM canola events were prepared. The limit of detection of the developed multiplex PCR was approximately 0.0125% for 3 GM canola events. Certified GM canola and stacked events were analyzed to validate the developed multiplex PCR. This study focuses on establishing multiplex PCR coupled with capillary electrophoresis for newly approved GM canola events and contributes to efficient monitoring GM canola samples in Korea.

Keywords: Genetically modified canola, Event-specific detection, Multiplex polymerase chain reaction, Monitoring, Capillary electrophoresis

Introduction

Nowadays, the production of Genetically modified (GM) crops on modern society is increasingly expanded. Compared with 1996, the area of GM crops in 2019 increased 113 times from 1.7 million hectares to 119.7 million hectares (James, 2019). The GM crops that dominate the cultivated area are soybean, maize, cotton, and canola (James, 2019). Among these 4 major GM crops, canola (Brassica L. napus) have been planted to produce cooking oil, livestock feed, and biodiesel fuel (KBCH 2018). As growing GM canola cultivated area, it is highly probable that unintended release of canola is into the natural environment during distribution process (Levina et al., 2019). Most GM canola events are herbicide-tolerant and pesticide-tolerant, ensuring high productivity. Farmers who adopted GM canola have gained economic profit (Brookes and Barfoot, 2014). However, because of GM traits, the impact of GM crops in the natural environment was significantly considered (Raman, 2017). Sharp disagreement over the effect of GMOs on the natural environment and human has occurred for decades (Tsatsakis et al., 2017). Therefore, many countries have implemented mandatory labeling system for GM foods (Zeng et al., 2021). For instance, European Union (EU) has enforced zero-tolerance policy on non-authorized GM material in feed (EU Commission Regulation 619/2011, 2011). There is an increasing necessity for efficient detection strategies to maintain strict regulations on the inflow of GM crops around the world (Park et al., 2017).

By August 2020, 7 crops (soybean, maize, cotton, canola, alfalfa, sugar-beet, and potato) and 177 GM events have been listed as obtaining authorization from Korean government (MFDS 2020). The Ministry of Food and Drug Safety (MFDS) in Korea has confirmed the 11 GM canola single events and 6 GM canola stacked events for food use in Korea. Over the last 5 years, 3 GM canola events (DP-073496-4, MON88302, and MS11) have been newly added to the approval list (MFDS 2020). To detect GM crops, numerous approaches have been established in many countries. Detection methods for GM events have been developed using DNA molecule and protein, which include conventional PCR, real-time PCR digital droplet PCR, loop-mediated isothermal amplification (LAMP), next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), and lateral flow strip (Fraiture et al., 2018; Gašparič et al., 2010; Köppel and Bucher, 2015; Liu et al., 2004, 2020; Zimmermann et al., 1999). Of these methods, PCR based on DNA has been typically used for GMO analysis (Park et al., 2017). Kim et al. (2015) demonstrated two pentaplex PCR assays for GM canola events (GT73, TOPAS 19/2, T45, MS1, MS8, RF1, RF2, and RF3). Also, Li et al. (2017) developed real-time PCR targeting 8 GM canola events (MS1, MS8, Oxy235, RT1, RF2, RT73, Topas 19/2, and T45). However, it still remains a need for detection method of newly approved GM canola events (DP-073496-4, MON88302, and MS11) in Korea after 2015.

Thus, this study aims to develop multiplex PCR detection method coupled with capillary electrophoresis for 3 approved GM canola events and contributes to improve monitoring system of imported canola in Korea.

Materials and methods

Sample preparation

Certified reference materials (CRM) of GM soybean (A2704-12, RRS, and MON89788), GM maize (MON810, T25, TC1507, NK603, and DAS-59122-7), GM cotton (MON1445 and MON531), GM canola (MS1, RF1, RF2, Topas 19/2, DP-073496-4, and MON88302) and non-GM canola samples were obtained from the American Oil Chemist’s Society (AOCS, Urbana, IL, USA) and the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium). GM Canola (GT73, MS8, MS11, RF3, and T45) and stacked events (DP-073496-4 × RF3, MON88302 × RF3, MON88302 × MS8 × RF3, MS11 × RF3, and MS11 × RF3 × MON88302) were kindly provided by the developers.

DNA extraction

Genomic DNA was extracted from CRMs using a DNeasy Plant Mini kit (Qiagen GmbH, Hilden, Germany) and FAST Genomic DNA Extraction kit (Pinucle, Yongin, Korea) following the manufacturer’s instruction with minor modification. The concentration and purity of extracted DNA were measured by using a Nano Spectrophotometer (Mastrogen, Las Vegas, NV, USA).

Designing primer for multiplex PCR

Event-specific primers were designed using the Primer Designer program, version 3.0 (Scientific and Educational Software, Durham, NC, USA). The primer sets 73496_F15/R15 and MON88302_F13/R9 were designed on the flanking sequence of DP-073496-4 (US Patent No. US8581046B2) and MON88302 (US Patent No. 20150119248A1), respectively. The primer set SHA086/MDB371_1 was designed based on the Korean Food Standards Codex of the MFDS. Detailed position of the primer designed was shown in Fig. 1. Cruciferin A gene (Gene accession no. X14555) was determined as a canola endogenous reference gene and the primer set MDB510/MDB511 was derived from the GMOMETHODS database of European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) (Jacchia et al. 2020). The information of primer used in this study was detailed in Table 1. All primers used in this study were synthesized by Bionics (Seoul, Korea).

Fig. 1.

Fig. 1

Open in a new tab

Schematic diagrams of event-specific primers designed to detect 3 GM canola events. The locations of the primers used in the multiplex PCR assays are indicated by arrows. All primer pairs were designed based on sequences of the transferred DNA flanking region and the inserted region of the GMO genome. P, promoter; T, terminator; L, RB, right border region; LB, left border region

Table 1.

Oligonucleotide primer pairs used in GM canola multiplex PCR assays

Targets Primer name Sequences (5′-3′) Length (bp) References
Cruciferin A gene MDB510 GGC CAG GGT TTC CGT GAT 101 Jacchia et al. 2019
MDB511' CCG TCG TTG TAG AAC CAT TGG
MS11 SHA086 CAA GAT GGG AAT TAA CAT CTA CAA ATT G 124 This study
MDB371_1 GAA ATC CAT GTA AAG CAG CAG
DP-073496–4 73496_F15 TGT TCG TTG GAG TTG TCT AC 170 This study
73496_R15 CGG TCC TAG ATC ATC AGT TC
MON88302 MON88302_F13 AGA TTG TAA CAC ACG GTT TCC TAC 223 This study
MON88302_R9 AGC AGG ACC TGC AGA AGC TTG
Open in a new tab

PCR conditions

Singleplex PCR and multiplex PCR were conducted by using a thermal cycler (PC808; ASTEC, Kyoto, Japan). Reaction components in 25 µL mixture for singleplex PCR were described as follows: 1X buffer (Bioneer, Daejeon, Korea), 10 mM of dNTP mix (2.5 mM of each dNTP), 0.4 µM of each primer, 0.5 unit of HotStart Taq polymerase, and 50 ng of genomic DNA. For multiplex PCR, reaction components composed of 1X buffer (Bioneer), 10 mM of dNTPs (2.5 mM of each dNTP), 1.0 unit of HotStart Taq polymerase, and 50 ng of genomic DNA. Final concentrations of primers for multiplex PCR were optimized as follows: 0.08 µM of MDB510/MDB511, 0.52 µM of SHA086/MDB371_1, 0.6 µM of 73,496 F15/R15, and 0.16 µM MON88302 F13/R9. The final reaction volume for both PCR was 25 µL. Singleplex PCR was performed as follow: pre-incubation at 94 °C for 5 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 30 s, and post-extension at 72 °C for 7 min. Multiplex PCR was performed with extension at 72 °C for 10 s. Amplified results confirmed by Agilent 2100 bioanalyzer with a DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA).

Assessment of sensitivity

A sensitivity of developed multiplex assay was determined using various levels of GM Canola DNA (DP-073496-4, MON88302, and MS11) mixed in non-GM canola DNA. The final concentration of mixture is composed of 1.25, 0.75, 0.25, 0.125, 0.025, and 0.0125% (w/w) for GM canola event, respectively.

Results and discussion

Specificity result of primers

Each event-specific primer was designed on the integration border region between the inserted gene and host genome. To verify specificity of the designed primers, various GM crops of GM soybean (A2704-12, RRS, and MON89788), GM maize (MON810, T25, TC1507, NK603, and DAS-59122-7), GM cotton (MON1445 and MON531), and GM canola (GT73, MS1, MS8, RF1, RF2, RF3, T45, Topas 19/2, DP-073496-4, MON88302, and MS11) were used in this study. As shown in Fig. 2, each primer for 3 GM canola events (DP-073496-4, MON88302, and MS11) specifically amplified target GM canola event with exact amplicon size and there was no any cross-amplification with other GM crops or non-target GM canola events.

Fig. 2.

Fig. 2

Open in a new tab

Specificity analysis of the 3 event-specific primer pairs. Lane L, marker (100-bp DNA ladder); lanes 1–11, GM canola (GT73, MS1, MS8, RF1, RF2, RF3, T45, Topas 19/2, DP-073496–4, MON88302, and MS11); lanes 12–14, GM soybean (RRS, MON89788, and A2704-12); lanes 15–19, GM maize (MON810, NK603, TC1507, T25, and DAS59122); lanes 20–21, GM cotton (MON1445 and MON531); lane 22, No template

Specificity and sensitivity results of multiplex PCR

In this study, multiplex PCR was established to detect 3 GM canola events (DP-073496-4, MON88302, and MS11) and Cruciferin A gene as an endogenous gene. For multiplex PCR, amplicon size of primer was determined with more than 20 base pairs intervals to make clearly distinguish multiple bands. Also, to obtain specific PCR band without non-specific PCR product and improve sensitivity in multiplex PCR, PCR condition and primer concentration were optimized. The optimized multiplex PCR showed each event-specific primer simultaneously generated multiple target sequence without cross-reactivity among targets (Fig. 3). In addition, the sensitivity of multiplex PCR was evaluated using various concentrations of DNA mixtures containing 1.25, 0.75, 0.25, 0.125, 0.025, and 0.0125% (w/w) of GM canola DNA in non-GM canola DNA. As shown in Fig. 4, the multiplex PCR developed in this study was capable of detecting up to 0.0125%. In Korea, GMO labeling legislation specifies 3% threshold for adventitious presence (MFDS notification 2019-98, 2019). Thus, it can be inferred that detection limit of the multiplex PCR is appropriate for the identification of the 3 approved GM canola events in Korea.

Fig. 3.

Fig. 3

Open in a new tab

Specific analysis of the multiplex PCR detection assays. Lane L, marker (100-bp DNA ladder); lane P, GM canola events MS11, DP-073496-4, MON88302, and endogenous gene; lane 1, GM canola event MS11 and endogenous gene, lane 2, GM canola event DP-073496–4 and endogenous gene, lane 3, GM canola event MON88302 and endogenous gene; lane N, no template

Fig. 4.

Fig. 4

Open in a new tab

Sensitivity analysis of the multiplex PCR detection method. Gel image (A) and electropherograms (B). Lane L, marker (100-bp DNA ladder); lanes (1)–(6), 1.25, 0.75, 0.25, 0.125, 0.025, and 0.0125% (w/w) for each GM event; lane N, no template. DNA mixture was prepared from 3 GM canola events (DP-073496-4, MON88302, and MS11) and non-GM canola at various levels

Validation of multiplex PCR in stacked GM Canola

To validate the developed multiplex PCR, 7 stacked GM canola events (DP-073496–4 × RF3, MON88302 × RF3, MON88302 × MS8 × RF3, MS11 × RF3, MS11 × RF3 × MON88302, MS8 × RF 3, and MS8 × RF3 × GT73) were investigated, which included non-target GM canola events such as RF3, MS8, and GT73 as well as 3 target events. 4 staked GM canola samples containing one target event were successfully amplified by each event-specific primer despite the presence of another GM canola event (lanes 1–4 in Fig. 5), and GM canola MS11 and MON88302 were simultaneously detected in the multiplex PCR (lane 5 in Fig. 5). Also, stacked GM canola samples without MS11, DP-073496-4, or MON88302 were not amplified by event-specific primers (lanes 6–7 in Fig. 6). Furthermore, an endogenous gene as internal control was detected in all stacked GM canola samples (Fig. 6). Therefore, we confirmed the multiplex PCR developed in this study correctly amplified only 3 GM canola events through validation analysis.

Fig. 5.

Fig. 5

Open in a new tab

The results of validation for stacked canola events using multiplex PCR. Lane L, 100 bp DNA ladder; lane P, Positive control; lane 1, DP-073496-4 × RF3, lane 2, MON88302 × RF3; lane 3, MON88302 × MS8 × RF3; lane 4, MS11 × RF3; lane 5, MS11 × RF3 × MON88302; lane 6, MS8 × RF3; lane 7, MS8 × RF3 × GT73; lane N, no template

This study indicates a simultaneous detection of 3 GM Canola events (DP-073496-4, MON88302, and MS11). We confirmed specificity and sensitivity of the multiplex PCR coupled with capillary electrophoresis. In order to verify developed method, 7 stacked GM canola events were analyzed. We represent a new approach to track authorized GM canola in Korea. To our knowledge, this detection method including MS11 event is first reported. This developed multiplex PCR can attribute to efficiently monitor GM canola events imported in Korea.

Acknowledgements

This work was supported by a grant from the Next-Generation BioGreen 21 Program (Project No. PJ01451101), Rural Development Administration, Republic of Korea.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Do-Geun Lee, Email: dob9395@naver.com.

Ji-Eun Park, Email: cr941213@gmail.com.

Mi-Ju Kim, Email: mijukim79@gmail.com.

Hyun-Joong Kim, Email: ritwwhjk@mokpo.ac.kr.

Hae-Yeong Kim, Email: hykim@khu.ac.kr.

References

  1. Brookes G, Barfoot P. Economic impact of GM crops: the global income and production effects 1996–2012. GM Crops & Food. 2014;5:65–75. doi: 10.4161/gmcr.28098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. European Union. COMMISSION REGULATION (EU) No 619/2011. Available from: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32011R0619. Accessed August. 10, 2020.
  3. Fraiture MA, Saltykova A, Hoffman S, Winand R, Deforce D, Vanneste K, Keersmaecke SCJD, Roosens NHC. Nanopore sequencing technology: a new route for the fast detection of unauthorized GMO. Scientific Reports. 2018;8:7903. doi: 10.1038/s41598-018-26259-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gašparič MB, Tengs T, Paz JLL, Jensen AH, Pla M, Esteve T, Zel J, Gruden K. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Analytical and Bioanalytical Chemistry. 2010;396:2023–2029. doi: 10.1007/s00216-009-3418-0. [DOI] [PubMed] [Google Scholar]
  5. Jacchia S, Sacco MG, Savini C, Mazzara M, Emons H. Event-specific Method for the Quantification of Oilseed Rape MS11 Using Real-time PCR Validation Report. Available from: https://gmo-crl.jrc.ec.europa.eu/summaries/EURL-VL-03-16-VR.pdf. Accessed August. 10, 2020.
  6. James C. Global Status of Commercialized Biotech/GM Crops in 2018. 10. Ithaca, NY, USA: ISAAA; 2019. [Google Scholar]
  7. Joint Research Centre. GMOMETHODS. European union reference laboratory for GM food and feed (EU-RL GMFF). Available from: http://gmo-crl.jrc.ec.europa.eu/gmomethods/. Accessed August. 10, 2020.
  8. Kim JH, Park SB, Hong YW, Kim HY. Detection of eight genetically modified canola events using two event-specific pentaplex PCR systems. Food Control. 2015;51:183–189. doi: 10.1016/j.foodcont.2014.11.027. [DOI] [Google Scholar]
  9. Köppel R, Bucher T. Rapid establishment of droplet digital PCR for quantitative GMO analysis. European Food Research and Technology. 2015;241:427–439. doi: 10.1007/s00217-015-2475-1. [DOI] [Google Scholar]
  10. Trend Report- Major GM crops.Korea Biosafety Clearing House (KBCH). Trend Report- Major GM crops. Available from: https://www.biosafety.or.kr/boardCnts/view.do?boardID=170&boardSeq=2056394&lev=0&m=040201&searchType=null&statusYN=W&page=1&s=kbch. Accessed August. 10, 2020.
  11. Levina LG, Levina B, Evelone B, Berga S, Kovalcuka L, Bergspica L, Jakovele A, MalisevsA VO, Rodze L, Rostoks N. Potential risk evaluation for unintended entry of genetically modified plant Propagating material in Europe through import of seeds and animal feed—the experience of Latvia. GM Crops & Food. 2019;10:159–169. doi: 10.1080/21645698.2019.1638721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Li Z, Li X, Wang C, Song G, Pi L, Zheng L, Zhang D, Yang L. One Novel multiple-target plasmid reference molecule targeting eight genetically modified canola events for genetically modified canola detection. Journal of Agricultural and Food Chemistry. 2017;65:8489–8500. doi: 10.1021/acs.jafc.7b02453. [DOI] [PubMed] [Google Scholar]
  13. Liu G, Su W, Xu Q, Long M, Zhou J, Song S. Liquid-phase hybridization based PCR-ELISA for detection of genetically modified organisms in food. Food Control. 2004;15:303–306. doi: 10.1016/S0956-7135(03)00081-1. [DOI] [Google Scholar]
  14. Liu H, Wang J, Li P, Bai L, Jia J, Pan A, Long X, Cui W, Tang X. Rapid detection of P–35S and T-nos in genetically modified organisms by recombinase polymerase amplification combined with a lateral flow strip. Food Control. 2020;107:106775. doi: 10.1016/j.foodcont.2019.106775. [DOI] [Google Scholar]
  15. Ministry of Food and Drugs Safety (MFDS). Korean Food Standards Codex (KFSC). Available from: http://foodsafetykorea.go.kr/foodcode/01_03.jsp?idx=786. Accessed August. 10, 2020.
  16. Ministry of Food and Drugs Safety (MFDS). Labeling Standards for Genetically Modified Foods. Available from: https://www.mfds.go.kr/eng/brd/m_15/view.do?seq=72429. Accessed August. 10, 2020.
  17. Ministry of Food and Drugs Safety (MFDS). Status of approval for safety review genetically modified foods. Available from: https://www.foodsafetykorea.go.kr/portal/board/board.do?menu_grp=MENU_NEW01&menu_no=2812. Accessed August. 10, 2020.
  18. Park SB, Kim HY, Kim JH. Detection of a screening method for the monitoring of 38 genetically modified maize events in food and feed in South Korea. Food Control. 2017;73:1459–1465. doi: 10.1016/j.foodcont.2016.11.009. [DOI] [Google Scholar]
  19. Raman R. The impact of genetically modified (GM) crops in modern agriculture: a review. GM Crops & Food. 2017;8:195–208. doi: 10.1080/21645698.2017.1413522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tsatsakis AM, Nawaz MA, Tutelyan VA, Golokhvast KS, Kalantzi OI, Chung DH, Kang SJ, Coleman MD, Tyshko N, Yang SH, Chung GH. Impact on environment, ecosystem, diversity and health from culturing and using GMOs as feed and food. Food and Chemical Toxicology. 2017;107:108–121. doi: 10.1016/j.fct.2017.06.033. [DOI] [PubMed] [Google Scholar]
  21. Zeng H, Wang J, Jia J, Wu G, Yang Q, Liu X, Tang X. Development of a lateral flow test strip for simultaneous detection of BT-Cry1Ab, BT-Cry1Ac and CP4 EPSPS proteins in genetically modified crops. Food Chemistry. 2021;335:127627. doi: 10.1016/j.foodchem.2020.127627. [DOI] [PubMed] [Google Scholar]
  22. Zimmermann A, Luthy J, Pauli U. Event specific transgene detection in Bt11 corn by quantitative PCR at the integration site. LWT - Food Science and Technology. 1999;33:210–216. doi: 10.1006/fstl.2000.0637. [DOI] [Google Scholar]

Articles from Food Science and Biotechnology are provided here courtesy of Springer

RESOURCES