Skip to main content
SpringerLink
Log in

Development of an Event-Specific Detection Method for Genetically Modified Maize IE034 by Quantitative Real-Time PCR

  • Published:
Food Analytical Methods Aims and scope Submit manuscript

Abstract

The insect-resistant transgenic maize event IE034 has been proved to be one of the most commercially developed transgenic maize events in China. This study was aimed to develop a stable and reliable quantitative detection method to monitor this new transgenic maize event. Here, we developed a novel event-specific real-time PCR method for this genetically modified maize event IE034. The resulting 134 base pair (bp) amplicon was designed according to the 5′ junction of inserted sequence and flanking maize genome sequence. Standard curve of the IE034 5′ event-specific sequence showed good linear regression and high PCR efficiency when using the IE034 pure line samples as calibrator. The limit of detection (LOD) for the IE034 detection method was estimated at approximately 8 initial template copies, and the limit of quantification (LOQ) was estimated at about 40 copies. The accuracy of this quantitative real-time PCR method was verified by screening four mixed DNA samples with known levels of the IE034 event (5, 1, 0.5, and 0.23 %, respectively). The quantified biases deviated from 8.7 to −12.2 %, and the relative standard deviation (RSD) ranged from 2.7 to 12.7 %. These data indicated that this new-developed IE034 event-specific real-time PCR method is suitable and reliable for the quantification of IE034 maize and its derivates.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Reference

  • Ahmed FE (2002) Detection of genetically modified organisms in foods. Trends Biotechnol 20(5):215–223

    Article  CAS  Google Scholar 

  • Bennett MD, Leitch IJ Plant DNA C-values Database of The Royal Botanic Garden. Kew, UK. http://www.rbgkew.org.uk/cval/exhibition/html/homepage.html

  • Buh Gašparič M, Tengs T, La Paz JL, Holst-Jensen A, Pla M, Esteve T, Žel J, Gruden K (2010) Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Anal Bioanal Chem 396(6):2023–2029

    Article  Google Scholar 

  • Chief Medical Officer of the Russian Federation (2007) Sanitary-epidemiological rules SanPiN 2.3.2.2227-07, additions and changes #5 to SanPiN 2.3.2.1078-01 “Hygiene Requirements to Safety and Nutrition Value of Food Products”. The resolution of the chief medical officer of the Russian Federation of June 25 2007, # 42. <http://www.fas.usda.gov/gainfiles/200707/146291830.pdf>

  • Choi SH (2011) Hexaplex PCR assay and liquid bead array for detection of stacked genetically modified cotton event 281-24-236×3006-210-23. Anal Bioanal Chem 401(2):647–655

    Article  CAS  Google Scholar 

  • Corbisier P, Bhat S, Partis L, Dan VR, Kerry X, Emslie R (2010) Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction. Anal Bioanal Chem 396(6):2143–2150

    Article  CAS  Google Scholar 

  • ENGL (2015) Definition of minimum performance requirements for analytical methods of GMO testing. Available at: http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm

  • European Food Safety Authority (2009). Topics A–Z. <http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1178621456978.htm>. Cited 23 Apr 2009

  • Guo J, Chen L, Liu X, Gao Y, Zhang D, Yang L (2012) A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs. Food Chem 132(3):1566–1573

    Article  CAS  Google Scholar 

  • Hernández M, Esteve T, Prat S, Pla M (2004) Development of real-time PCR system based on SYBR Green I, Amplifluor and TaqMan technologies for specific quantitative detection of the transgenic maize event GA21. J Cereal Sci 39:99–107

    Article  Google Scholar 

  • Holst-Jensen A, Røning SB, Løseth A, Berdal KG (2003) PCR technology for screening and quantification of genetically modified organisms (GMOs). Anal Bioanal Chem 375:985–993

    Article  CAS  Google Scholar 

  • James C (2015) 20th Anniversary (1996 to 2015) of the global commercialization of biotech crops and biotech crop highlights in 2015. ISAAA Brief No. 51. ISAAA, Ithaca

    Google Scholar 

  • James C (2014) Global Status of Commercialized Biotech/GM Crops. International Service for the Acquisition of Agri-Biotech Applications (ISAAA), Ithaca.

  • Kamle S, Ali S (2013) Genetically modified crops: detection strategies and biosafety issues. Gene 522(2):123–132

    Article  CAS  Google Scholar 

  • Koressaar T, Remm M (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23(10):1289–1291

    Article  CAS  Google Scholar 

  • Li P, Jia J, Bai L, Pan A, Tang X (2013) Identification and quantification of genetically modified moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods. Appl Biochem Biotechnol 170(5):1151–1162

    Article  CAS  Google Scholar 

  • Li F., Yan W., Long L., Xu J., Liu Y., Li C., Dong L., Wu F. (2014a) Specific quantitative PCR primers, probes and method of GM maize IE034 event. CN patent. 2014195257 [P]. 2014–12-10

  • Li F., Long L., Li C., Xu, J., Liu, Y., Yan, W., Dong L., Wu, F. (2014b) Qualitative detection PCR primers, assay and kit of GM maize IE034 event. CN patent. 2014195256A [P]. 2014–12-10

  • Li X, Wang X, Yang J, Liu Y, He Y, Pan L (2014c) A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40). BMC Biotechnol 14:43

    Article  Google Scholar 

  • Matsuoka T (2001) GMO labeling and detection methods in Japan. APEC-JIRCAS joint symposium and workshop on agricultural biotechnology

  • Ministry of Agriculture and Forestry of South Korea (2000). Guidelines for labeling of genetically modified agricultural products. MAF notification, 31

  • Ministry of Agriculture, 1782 announcement-8-2012, detection of genetically modified plants and derived products-technical specification for manufacture of matrix reference material. China Agriculture Press, Beijing

  • Miraglia M, Berdal KG, Brera C, Corbisier P, Holst-Jensen A, Kok EJ et al (2004) Detection and traceability of genetically modified organisms in the food production chain. Food Chem Toxicol 42:1157–1180

    Article  CAS  Google Scholar 

  • Nielsen CR, Berdal KG, Holst-Jensen A (2004) Characterisation of the 5′ integration site and development of an event-specific real-time PCR assay for NK603 maize from a low starting copy number. Eur Food Res Technol 219(4):421–427

    Article  CAS  Google Scholar 

  • Pan AH, Yang LT, Xu SC, Yin CS, Zhang KW, Wang ZY et al (2006) Event specific qualitative and quantitative PCR detection of MON863 maize based upon the 30-transgene integration sequence. J Cereal Sci 43:250–257

    Article  CAS  Google Scholar 

  • Raymond Park J, McFarlane I, Hartley Phipps R, Ceddia G (2011) The role of transgenic crops in sustainable development. Plant Biotechnol J 9(1):2–21

    Article  Google Scholar 

  • Regulation (EC) No 619 (2011) of 24 June 2011 laying down the methods of sampling and analysis for the official control of feed as regards presence of genetically modified material for which an authorization procedure is pending or the authorization of which has expired. Off. Eur. Union. 2011,L166,9–15

  • Regulation (EC) No. 1829 (2003) of the European parliament and of the council of 22 September 2003 on genetically modified food and feed [Official Journal L 268/1 of 18.10.2003]

  • Serageldin I (1999) Biotechnology and food security in the 21st century. Science 285:387–389

    Article  CAS  Google Scholar 

  • Takabatake R, Onishi M, Koiwa T, Futo S, Minegishi Y, Akiyama H, Teshima R, Furui S, Kitta K (2010) Establishment and evaluation of event-specific quantitative PCR method for genetically modified soybean MON89788. Shokuhin Eiseigaku Zasshi 51(5):242–246

    Article  CAS  Google Scholar 

  • Wang G, Liu Y, Zhang Y, Liu Y (2012) Flanking sequence of foreign insert segment in maize genetic modification event IE034 and application of flanking sequence. China Patent CN 102604940 B [P].2012–7-25

  • Wu G, Wu Y, Xiao L, Lu C (2009) Event-specific qualitative and quantitative PCR detection of genetically modified rapeseed Topas 19/2. Food Chem 112:232–238

    Article  CAS  Google Scholar 

  • Yang L, Xu S, Pan A, Yin C, Zhang K, Wang Z, Zhou Z, Zhang D (2005) Event-specific qualitative and quantitative PCR detection of genetically modified MON863 maize based on the 5-transgene integration sequence. J Agric Food Chem 53:9312–9318

    Article  CAS  Google Scholar 

  • Yang R, Xu WT, Luo YB, Guo F, Lu Y, Huang KL (2007) Event specific qualitative and quantitative polymerase chain reaction detection of roundup ready event GT73 based on the 30-transgene integration sequence. Plant Cell Repots 26:1821–1831

    Article  CAS  Google Scholar 

  • Yang C, Zhang D, Yang L (2013) Development of event-specific PCR detection methods for genetically modified tomato Huafan no. 1. J Sci Food Agric 93(3):652–660

    Article  CAS  Google Scholar 

  • Zhang M, Huo N, Liu Y, Qiu Y, Ao J, Luan F, Li Q, Gao X (2012) Event-specific detection of genetically modified wheat B73-6-1 based on the 3′-flanking sequence. Eur Food Res Technol 235:1149–1159

    Article  CAS  Google Scholar 

  • Zhang Y, Liu Y, Ren Y, Liu Y, Liang G, Song F, Bai S, Wang J, Wang G (2013) Overexpression of a novel Cry1Ie gene confers resistance to Cry1Ac-resistant cotton bollworm in transgenic lines of maize. Plant Cell Tiss Organ Cult 115:151–158

    Article  CAS  Google Scholar 

  • Zhang L, Huang X, Zhu S (2014) An event-specific real-time PCR detection system for the transgenic rice line 114-7-2 of producing functional human serum albumin. Eur Food Res Technol 239:403–408

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. College of Plant Science, Jilin University, Changchun, 130062, China

    Feiwu Li & Shihong Zhang

  2. Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences, Changchun, 130033, China

    Feiwu Li, Likun Long, Wei Yan & Congcong Li

  3. Institute of Quality Standards for Agricultural Products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China

    Junfeng Xu

  4. Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Yunjun Liu

Authors
  1. Feiwu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Likun Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Congcong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Junfeng Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yunjun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shihong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shihong Zhang.

Ethics declarations

Funding

This work was supported by the National Transgenic Plant Special Fund (China) (2014ZX08012-001), Jilin Agricultural Science and Technology Innovation Program (2013). We do appreciate the financial support, and the authors acknowledge the financial support for this study.

Conflict of Interest

Feiwu Li declares that he has no conflict of interest. Likun Long declares that she has no conflict of interest. Wei Yan declares that she has no conflict of interest. Congcong Li declares that he has no conflict of interest. Junfeng Xu declares that he has no conflict of interest. Yunjun Liu declares that he has no conflict of interest. Shihong Zhang declares that he has no conflict of interest. IE034 event-specific sequence had been approved to use by the inventor of the patent (China patent no. CN 102604940 B).

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Not applicable

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, F., Long, L., Yan, W. et al. Development of an Event-Specific Detection Method for Genetically Modified Maize IE034 by Quantitative Real-Time PCR. Food Anal. Methods 10, 1727–1735 (2017). https://doi.org/10.1007/s12161-016-0707-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12161-016-0707-z

Keywords

Search

Navigation