European Food Research and Technology

, Volume 242, Issue 8, pp 1277–1284 | Cite as

Development of event-specific qualitative and quantitative PCR detection methods for the transgenic maize BVLA430101

  • Jun Rao
  • Litao Yang
  • Jinchao Guo
  • Sheng Quan
  • Guihua Chen
  • Xiangxiang Zhao
  • Dabing Zhang
  • Jianxin Shi
Original Paper


The transgenic maize (Zea mays L.) line BVLA430101 overexpressing Aspergillus niger phyA2 was approved in 2009 to be planted in a given area in China. However, the flanking sequences and event-specific qualitative/quantitative PCR detection methods for this transgenic event have not been reported. In this study, we characterized the molecular features of the exogenous integration in BVLA430101 and developed event-specific qualitative/quantitative PCR detection methods. Using genome walking, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), and conventional PCR assays, we revealed that one intact copy of phytase construct was integrated in the maize genome chromosome 3, which is followed by a fragment of the transformation vector containing partial sequence of pH2b promoter. The defined 3′ flanking sequence was 1644 bp in length, based on which the event-specific qualitative and quantitative PCR assays for BVLA430101 were developed. The limit of detection (LOD) of the qualitative PCR assay was 10 haploid genome copies, and the limits of detection and quantification (LOD and LOQ) of the quantitative PCR were 10 and 10 copies of maize haploid genome, respectively. In-house validation of the developed event-specific quantitative PCR method with three practical maize samples showed that the quantified biases between the test and true values ranged between 2.60 and 7.52 %. These results indicated that the developed event-specific qualitative and quantitative PCR methods based on the newly characterized 3′ flanking sequences can be used successfully for the identification and quantification of transgenic maize BVLA430101 and its derived products.


Integration flanking sequence Limit of detection Limit of quantification Phytase Qualitative and quantitative PCR Transgenic maize BVLA430101 



We thank Dr. Rumei Chen from China Academy of Agricultural Sciences for supplying the seeds of transgenic maize BVLA430101 and its non-transgenic counterpart used in the present study.

Funding sources

This work was supported by the China National Transgenic Plant Special Fund (2013ZX08012-002 and 2014ZX08012-002), China Innovative Research Team, Ministry of Education, and the Programme of Introducing Talents of Discipline to Universities (111 Project, B14016), and Shanghai Leading Talent Project in Agriculture.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Compliance with ethics requirements

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee. This article does not contain any studies with animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

217_2015_2631_MOESM1_ESM.docx (35 kb)
Supplementary material 1 (DOCX 34 kb)


  1. 1.
    James C (2014) Global status of commercialized biotech/GM crops: 2014. ISAAA brief no. 49. ISAAA, IthacaGoogle Scholar
  2. 2.
    Dlugosch KM, Whitton J (2008) Can we stop transgenes from taking a walk on the wild side? Mol Ecol 17:1167–1169CrossRefGoogle Scholar
  3. 3.
    Zhang D, Guo J (2011) The development and standardization of testing methods for genetically modified organisms and their derived products. J Integr Plant Biol 53:539–551CrossRefGoogle Scholar
  4. 4.
    European Commission Regulation (EC) 1829/2003 and 1830/2003. Official J Eur Communities: Legislation 268:1–28Google Scholar
  5. 5.
    Carter CA, Gruere GP (2003) International approaches to the labeling of genetically modified foods. Choices 18:1–4Google Scholar
  6. 6.
    Ministry of Agriculture, the People’s Republic of China (2002) Biosafety assessment and regulation on genetically modified organisms. Order no. 8, BeijingGoogle Scholar
  7. 7.
    Angers-Loustau A, Petrillo M, Bonfini L, Gatto F, Rosa S, Patak A, Kreysa J (2014) JRC GMO-matrix: a web application to support genetically modified organisms detection strategies. BMC Bioinformatics 15:417CrossRefGoogle Scholar
  8. 8.
    Pi L, Li X, Cao Y, Wang C, Pan L, Yang L (2015) Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events. Anal Bioanal Chem 407:2877–2886CrossRefGoogle Scholar
  9. 9.
    Tam PD (2015) Genetically modified organism (GMO) detection by biosensor based on SWCNT material. Curr Appl Phys 15:397–401CrossRefGoogle Scholar
  10. 10.
    Barbau-Piednoir E, Stragier P, Roosens N, Mazzara M, Savini C, Van den Eede G, Van den Bulcke M (2014) Inter-laboratory testing of GMO detection by combinatory SYBR® green PCR screening (CoSYPS). Food Anal Method 7:1719–1728Google Scholar
  11. 11.
    Dobnik D, Spilsberg B, Bogožalec Košir A, Holst-Jensen A, Žel J (2015) Multiplex quantification of twelve EU authorized GM maize lines with droplet digital PCR. Anal Chem 87:8218–8226CrossRefGoogle Scholar
  12. 12.
    Guo J, Yang L, Liu X, Guan X, Jiang L, Zhang D (2009) Characterization of the exogenous insert and development of event-specific PCR detection methods for genetically modified Huanong No. 1 papaya. J Agric Food Chem 57:7205–7212CrossRefGoogle Scholar
  13. 13.
    Wu Y, Yang L, Cao Y, Song G, Shen P, Zhang D, Wu G (2013) Collaborative validation of an event-specific quantitative real-time PCR method for genetically modified rice event TT51-1 detection. J Agric Food Chem 61:5953–5960CrossRefGoogle Scholar
  14. 14.
    Liu Y, Zhang MH, Yu YB, Sun Z, Zhen Z, Gao XJ (2014) Event-specific qualitative and quantitative detection in transgenic soybean OsDREB3 based on the 5′ flanking sequence. Food Biotechnol 28:63–78CrossRefGoogle Scholar
  15. 15.
    Jacchia S, Nardini E, Bassani N, Savini C, Shim JH, Trijatmiko K, Kreysa J, Mazzara M (2015) International ring trial for the validation of an event-specific Golden Rice 2 quantitative real-time PCR method. J Agric Food Chem 63:4954–4965CrossRefGoogle Scholar
  16. 16.
    Wei J, Le H, Pan A, Xu JF, Li FW, Li X, Quan S, Guo JC, Yang LT (2015) Collaborative trial for the validation of event-specific PCR detection methods of genetically modified papaya Huanong No. 1. Food Chem 194:20–25CrossRefGoogle Scholar
  17. 17.
    Li Y, Peng Y, Hallerman EM, Wu K (2014) Biosafety management and commercial use of genetically modified crops in China. Plant Cell Rep 33:565–573CrossRefGoogle Scholar
  18. 18.
    Chen RM, Xue GX, Chen P, Yao B, Yang WZ, Ma QL, Fan YL, Zhao ZY, Tarczynski MC, Shi JR (2008) Transgenic maize plants expressing a fungal phytase gene. Transgenic Res 17:633–643CrossRefGoogle Scholar
  19. 19.
    Rao J, Yang L, Wang C, Zhang D, Shi J (2013) Digital gene expression analysis of mature seeds of transgenic maize overexpressing Aspergillus niger phyA2 and its non-transgenic counterpart. GM Crops Food 4:98–108CrossRefGoogle Scholar
  20. 20.
    Su C, Sun Y, Xie J, Peng Y (2011) A construct-specific qualitative and quantitative PCR detection method of transgenic maize BVLA430101. Eur Food Res Technol 233:117–122CrossRefGoogle Scholar
  21. 21.
    Huang X, Chen L, Xu J, Ji HF, Zhu S, Chen H (2014) Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification method. Food Chem 156:184–189CrossRefGoogle Scholar
  22. 22.
    Zhang FL, Song J, Niu B, Yin Q, Chang LJ, Wang D, Liu WJ, Lei SR, Liu Y (2015) An event-specific qualitative and real-time PCR detection of 98140 maize in mixed samples. Food Control 57:1–8CrossRefGoogle Scholar
  23. 23.
    Zhang Q, Chen RM, Yang WZ, Cheng P, Xue GX, Fan YL (2010) The obtaining of transgenic maize plants with phyA2 gene constitutive express phytase. J Agric Biotechnol 18:623–629Google Scholar
  24. 24.
    Pawlowski WP, Somers DA (1998) Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc Natl Acad Sci USA 95:12106–12110CrossRefGoogle Scholar
  25. 25.
    European Network of GMO Laboratories (ENGL) (2008) Definition of minimum performance requirements for analytical methods of GMO testing.

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Jun Rao
    • 1
    • 2
  • Litao Yang
    • 1
  • Jinchao Guo
    • 1
  • Sheng Quan
    • 1
    • 3
  • Guihua Chen
    • 1
  • Xiangxiang Zhao
    • 4
  • Dabing Zhang
    • 1
  • Jianxin Shi
    • 1
    • 3
  1. 1.Joint International Research Laboratory of Metabolic and Developmental Sciences, SJTU-University of Adelaide Joint Centre for Agriculture and Health, Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiChina
  2. 2.Jiangxi Cancer HospitalNanchangChina
  3. 3.Shanghai Ruifeng Agro-biotechnology Co., LtdShanghaiChina
  4. 4.Department of Life ScienceHuaiyin Normal CollegeHuaianChina

Personalised recommendations