, Volume 73, Issue 1, pp 21–29 | Cite as

Development of a multiplex fluorescence quantitative PCR for detection of genetically modified organisms

  • Fengjun Wang
  • Junli Feng
  • Sudan Ye
  • Hannian Huang
  • Xianglin Zhang
Original Article


The commercially available genetically modified plants authorized worldwide and therefore the target sequences for molecular detection of genetically modified organisms (GMOs) are ever-increasing. The European Union has implemented a set of very strict procedures for approval to grow, import and/or utilize GMOs as food or food ingredients. As a result, GMO laboratories and food production industry currently are forced to apply different methods to test raw material and complex processed food products. Three exogenous genes (the 35 s promoter of the cauliflower mosaic virus (35 s), nos terminator from Agrobacterium tumefaciens (nos), and the neomycin phosphotransferase II (nptII) gene) are commonly used in GMO detection. In this paper, a multiplex quantitative real-time PCR (qPCR) system was developed which allows simultaneously detection of the three exogenous genes in one reaction tube. The determined limits for the multiplex qPCR assays were 4 copies/reaction in maize samples. The specificity of the assays was demonstrated to be 100% according to the detection results of 23 genetically modified (GM) crops and 97 complex processed food products. The validation data show the individual PCR efficiency was accredited with negligible impacts between three detection channels in 7500 fluorescence quantitative PCR machine. These results indicate that this high-throughput multiplex qPCR method which combined with a reference gene is feasible for screening of GMOs, even for the processed food.


Detection Genetically modified organisms Multiplex qPCR Screening 



This work was supported by the Grant from the Science Research Project of Zhejiang Federation of Supply and Marketing Cooperative under grant (No. 16SSY04); the Science Research Project of Zhejiang Institute of Economic and Trade under grant (No. 16QN02).


  1. Ao JX, Li QZ, Gao XJ, Yu YB, Li L, Zhang MH (2011) A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean, maize and rice in highly processed products. Food Control 22:1617–1623CrossRefGoogle Scholar
  2. Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218CrossRefGoogle Scholar
  3. Bahrdt C, Krech AB, Wurz A, Wulff D (2010) Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed. Anal Bioanal Chem 396:2103–2112CrossRefPubMedGoogle Scholar
  4. Batista R, Oliveira MM (2009) Facts and fiction of genetically engineered food. Trends Biotechnol 27:277–286CrossRefPubMedGoogle Scholar
  5. Bucher TB, Fridez F, Koppel R (2014) Duplex real-time PCR for the determination of non-Basmati rice in Basmati rice (Oryza sativa). Eur Food Res Technol 238:417–423CrossRefGoogle Scholar
  6. Bustin SA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefPubMedGoogle Scholar
  7. Chaouachi M et al (2008) A high-throughput multiplex method adapted for GMO detection. J Agric Food Chem 56:11596–11606CrossRefPubMedGoogle Scholar
  8. Ciabatti I, Marchesi U, Froiio A, Paterno A, Ruggeri M, Amaddeo D (2005) Role of the "National Reference Centre for Genetically Modified Organisms (GMO) detection" in the official control of food and feed. Vet Res Commun 29(Suppl. 2):31–34CrossRefPubMedGoogle Scholar
  9. Costa TE, Marin VA (2011) Labeling of food containing genetically modified organisms: international policies and Brazilian legislation. Cien Saude Colet 16:3571–3582CrossRefPubMedGoogle Scholar
  10. Dorries HH, Remus I, Gronewald A, Gronewald C, Berghof–Jager K. (2010) Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs). Anal Bioanal Chem 396:2043–2054CrossRefPubMedGoogle Scholar
  11. Garson JA et al (2009) Unreliable Real-Time PCR Analysis of Human Endogenous Retrovirus-W (HERV-W) RNA Expression and DNA Copy Number in Multiple Sclerosis. AIDS Res Hum Retrovir 25:377–378CrossRefPubMedGoogle Scholar
  12. Gaudron T, Peters C, Boland E, Steinmetz A, Moris G (2009) Development of a quadruplex-real-time-PCR for screening food for genetically modified organisms. Eur Food Res Technol 229:295–305CrossRefGoogle Scholar
  13. Halpin C (2005) Gene stacking in transgenic plants--the challenge for 21st century plant biotechnology. Plant Biotechnol J 3:141–155CrossRefPubMedGoogle Scholar
  14. Holst-Jensen A, Ronning SB, Lovseth A, Berdal KG (2003) PCR technology for screening and quantification of genetically modified organisms (GMOs). Anal Bioanal Chem 375:985–993CrossRefPubMedGoogle Scholar
  15. James C (2015) 20th Anniversary (1996 to 2015) of the Global Commercialization of Biotech Crops and Biotech Crop Highlights in 2015. ISAAA Brief 51. ISAAA, IthacaGoogle Scholar
  16. Koppel R, Bucher T, Frei A, Waiblinger HU (2015) Droplet digital PCR versus multiplex real-time PCR method for the detection and quantification of DNA from the four transgenic soy traits MON87769, MON87708, MON87705 and FG72, and lectin. Eur Food Res Technol 241:521–527CrossRefGoogle Scholar
  17. Koppel R, Sendic A, Waiblinger HU (2014) Two quantitative multiplex real-time PCR systems for the efficient GMO screening of food products. Eur Food Res Technol 239:653–659CrossRefGoogle Scholar
  18. Li P et al (2012) Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5′-transgene integration sequence. Genet Mol Res 11:1117–1129CrossRefPubMedGoogle Scholar
  19. Marmiroli N et al (2008) Methods for detection of GMOs in food and feed. Anal Bioanal Chem 392:369–384CrossRefPubMedGoogle Scholar
  20. Randhawa GJ, Chhabra R, Singh M (2009) Multiplex PCR-based simultaneous amplification of selectable marker and reporter genes for the screening of genetically modified crops. J Agric Food Chem 57:5167–5172CrossRefPubMedGoogle Scholar
  21. Randhawa GJ, Sharma R, Singh M (2012) Qualitative and event-specific real-time PCR detection methods for Bt brinjal event EE-1. J AOAC Int 95:1733–1739CrossRefPubMedGoogle Scholar
  22. Randhawa GJ, Singh M (2012) Multiplex, construct-specific, and real-time PCR-based analytical methods for Bt rice with cry1Ac gene. J AOAC Int 95:186–194CrossRefPubMedGoogle Scholar
  23. Taverniers I, Papazova N, Bertheau Y, De Loose M, Holst-Jensen A (2008) Gene stacking in transgenic plants: towards compliance between definitions, terminology, and detection within the EU regulatory framework. Environ Biosaf Res 7:197–218CrossRefGoogle Scholar
  24. Waiblinger HU, Boernsen B, Pietsch K (2008) GMO routine analysis - Screening table for detection of genetically modified plants in food and feed. Deut Lebensmittel-Rundschau 104:261–264Google Scholar
  25. Zhang D, Guo J (2011) The development and standardization of testing methods for genetically modified organisms and their derived products. J Integr Plant Biol 53:539–551CrossRefPubMedGoogle Scholar
  26. Zhang X, Wang C, Chen Y, Zhang J, Zhang W (2013) Detection of genetically modified components--Tomato test methods. Industry standard of entry-exit inspection and quarantine of the People’s Republic of China, SN/T 1618. Industry Standard, ChinaGoogle Scholar
  27. Zhu SF, Tan W, Cao JJ, Zhang GM, Pan LW, Huang WS, Chen HY (2003) Protocol of the real-time PCR for detecting genetically modified plants and their derived products, industry standard of entry-exit inspection and quarantine of the People’s Republic of China. SN/T 1204. Industry Standard, ChinaGoogle Scholar

Copyright information

© Section Botany: © Plant Science and Biodiversity Centre, Slovak Academy of Sciences 2018

Authors and Affiliations

  • Fengjun Wang
    • 1
  • Junli Feng
    • 2
  • Sudan Ye
    • 1
  • Hannian Huang
    • 1
  • Xianglin Zhang
    • 3
  1. 1.Department of Applied EngineeringZhejiang Institute of Economic and TradeHangzhouPeople’s Republic of China
  2. 2.Institute of SeafoodZhejiang Gongshang UniversityHangzhouPeople’s Republic of China
  3. 3.Xinjiang Entry-Exit Inspection and Quarantine BureauUrumqiPeople’s Republic of China

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