Skip to main content

Advertisement

SpringerLink
Log in

Hexaplex PCR assay and liquid bead array for detection of stacked genetically modified cotton event 281-24-236×3006-210-23

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

A hexaplex system based on multiplex polymerase chain reaction (PCR) coupled with liquid bead array was developed to assist detection of stacked genetically modified (GM) cotton event 281-24-236 × 3006-210-23 (Widestrike) expressing two kinds of endotoxin from Bacillus thuringiensis (Bt). The efficiency of this multiplex detection system was assessed. Specific primer sets for simultaneous detection of six targets in the stacked GM cotton event were constructed and used for the PCR assay. Each of the six targets was amplified, and the amplicons could be separated as discrete bands by agarose gel electrophoresis. A liquid bead array assay for the stacked GM cotton was performed using the hexaplex PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescent signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescent intensity values. Comparison of the assays showed that results from the liquid bead array using specific probes agreed with those from the PCR, and detection of the different target elements was found to be very specific with no cross-reaction. Therefore, the combination of hexaplex PCR and liquid bead array for detection of stacked GM events can be a useful and efficient system for screening and analyzing multiple transgenes for simultaneous qualitative analysis.

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

References

  1. James C (2010) Global Status of Commercialized Biotech/GM Crops: 2010. ISAAA Brief No. 42. ISAAA: Ithaca, NY

  2. Akiyama H, Watanabe T, Wakabayashi K, Nakade S, Yasui S, Sakata K, Chiba R, Spiegelhalter F, Hino A, Maitani T (2005) Quantitative detection system for maize sample containing combined-trait genetically modified maize. Anal Chem 77:7421–7428

    Article  CAS  Google Scholar 

  3. Taverniers I, Windels P, Vaitilingom M, Milcamps A, Van Bockstaele E, Van den Eede G, De Loose M (2005) Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176, and GA21 maize and transgenic GT73 canola. J Agric Food Chem 53:3041–3052

    Article  CAS  Google Scholar 

  4. Lee SH, Kim JK, Yi BY (2007) Detection methods for biotech cotton MON 15985 and MON 88913 by PCR. J Agric Food Chem 55:3351–3357

    Article  CAS  Google Scholar 

  5. Baeumler S, Wulff D, Tagliana L, Song P (2006) A real-time quantitative PCR detection method specific to widestrike transgenic cotton (Event 281-24-236/3006-210-23). J Agri Food Chem 54:6527–6534

    Article  CAS  Google Scholar 

  6. Chaouachi M, Chupeau G, Berard A, McKhann H, Romaniuk M, Giancola S, Laval V, Bertheau Y, Brunel D (2008) A high-throughput multiplex method adapted for GMO detection. J Agric Food Chem 56:11596–11606

    Article  CAS  Google Scholar 

  7. De Schrijver A, Devos Y, Van den Bulcke M, Cadot P, De Loose M, Reheul D, Sneyers M (2007) Risk assessment of GM stacked events obtained from crosses between GM events. Trends Food Sci Technol 18:101–109

    Article  Google Scholar 

  8. Lu IJ, Lin CH, Pan TM (2010) Establishment of a system based on universal multiplex-PCR for screening genetically modified crops. Anal Bioanal Chem 396:2055–2064

    Article  CAS  Google Scholar 

  9. Querci M, Foti N, Bogni A, Kluga L, Broll H, Van den Eede G (2009) Real-time PCR-based ready-to-use multi-target analytical system for GMO detection. Food Anal Methods 2:325–336

    Article  Google Scholar 

  10. Shan G, Embrey SK, Schafer BW (2007) A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton. J Agric Food Chem 55:5974–5979

    Article  CAS  Google Scholar 

  11. Choi SH, Oh YT, Kwon JY, Lee SN, Han BD, Ryu KH (2010) Development of detection system using multiplex PCR and liquid beadarray for stacked genetically modified rice event (LS28 × Cry1Ac). J Korean Soc Appl Biol Chem 53:639–646

    Article  CAS  Google Scholar 

  12. Holst-Jensen A (2009) Testing for genetically modified organisms (GMOs): past, present and future perspectives. Biotech Adv 27:1071–1082

    Article  CAS  Google Scholar 

  13. Onishi M, Matsuoka T, Kodama T, Kashiwaba K, Futo S, Akiyama H, Maitani T, Furui S, Oguchi T, Hino A (2005) Development of a multiplex polymerase chain reaction method for simultaneous detection of eight events of genetically modified maize. J Agri Food Chem 53:9713–9721

    Article  CAS  Google Scholar 

  14. Schmidt A, Sahota R, Pope DS, Lawrence TS, Belton MP, Rott ME (2008) Detection of genetically modified canola using multiplex PCR coupled with oligonucleotide microarray hybridization. J Agric Food Chem 56:6791–6800

    Article  CAS  Google Scholar 

  15. Shrestha HK, Hwu KK, Wang SJ, Liu LF, Chang MC (2008) Simultaneous detection of eight genetically modified maize lines using a combination of event- and construct-specific multiplex-PCR technique. J Agric Food Chem 56:8962–8968

    Article  CAS  Google Scholar 

  16. Kim JH, Park SH, Kim HY (2009) Multiplex PCR detection of four events of GM maize (Event 3272, LY038, MIR162, and MON88017). J Korean Soc Appl Biol Chem 52:105–107

    Article  CAS  Google Scholar 

  17. Chiueh LC, Chen YL, Shih YC (2002) Study on the detection method of six varieties of genetically modified maize and processed foods. J Food Drug Anal 10:25–33

    CAS  Google Scholar 

  18. Kim JH, Kim TW, Lee WY, Park SH, Kim HY (2007) Multiplex PCR detection of the GT73, MS8 × RF3, and T45 varieties of GM canola. Food Sci Biotechnol 16:104–109

    Google Scholar 

  19. Quirasco M, Schoel B, Chhalliyil P, Faga J, Gálvez A (2008) Real-time and conventional PCR detection of Liberty Link® rice varieties and transgenic soy in rice sampled in the Mexican and American retail markets. Anal Bioanal Chem 392:395–404

    Article  CAS  Google Scholar 

  20. Xu J, Miao H, Wu H, Huang W, Tang R, Qiu M, Wen J, Zhu S (2006) Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray. Biosens Bioelectron 22:71–77

    Article  CAS  Google Scholar 

  21. Wilson WJ, Erler AM, Nasarabadi SL, Skowronski EW, Imbro PM (2005) A multiplex PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents. Mol Cell Probes 19:137–144

    Article  CAS  Google Scholar 

  22. Dunbar SA (2006) Application of Luminex® XMAPTM technology for rapid, high-throughput multiplexed nucleic acid detection. Clin Chim Acta 363:71–82

    Article  CAS  Google Scholar 

  23. Kim JH, Kim SY, Lee H, Kim YR, Kim HY (2010) An event-specific DNA microarray to identify genetically modified organisms in processed foods. J Agri Food Chem 58:6018–6026

    Article  CAS  Google Scholar 

  24. Oh YT, Park SW, Chun S, Lim N, Ahn KS, Ka J, Jin D, Han BD (2009) Genotyping of CYP21A2 for congenital adrenal hyperplasia screening using allele-specific primer extension followed by bead array hybridization. Mol Diagn Ther 13:397–405

    CAS  Google Scholar 

  25. Yang L, Chen J, Huang C, Liu Y, Jia S, Pan L, Zhang D (2005) Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons. Plant Cell Rep 24:237–245

    Article  CAS  Google Scholar 

  26. Randhawa GJ, Chhabra R, Singh M (2010) Decaplex and real-time PCR based detection of MON531 and MON15985 Bt cotton events. J Agri Food Chem 58:9875–9881

    Article  CAS  Google Scholar 

  27. Aslanian S, Azimi M, Noble J, Hoppe C (2007) Application of flow cytometry-based genotyping for rapid detection of hemoglobin variants. Int J Lab Hematol 29:284–291

    Article  CAS  Google Scholar 

  28. Nadal A, Esteve T (2009) Multiplex polymerase chain reaction-capillary gel electrophoresis: A promising tool for GMO screening-assay for simultaneous detection of five genetically modified cotton events and species. J AOAC Int 92:765–772

    CAS  Google Scholar 

  29. Mahony J, Chong S, Merante F, Yaghoubian S, Sinha T, Lisle C, Janeczko R (2007) Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a liquid beadarray-based assay. J Clin Microbiol 45:2965–2970

    Article  CAS  Google Scholar 

  30. Leimanis S, Hernandez M, Fernandez S, Boyer F, Burns M, Bruderer S, Glouden T, Harris N, Kaeppeli O, Philipp P, Pla M, Puigdomènech P, Vaitilingom M, Bertheau Y, Remacle J (2006) A microarray-based detection system for genetically modified (GM) food ingredients. Plant Mol Biol 61:123–139

    Article  CAS  Google Scholar 

  31. 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 229:295–305

    Article  CAS  Google Scholar 

  32. Waiblinger HU, Grohmann L, Mankertz J, Engelbert D, Pietsch K (2010) A practical approach to screen for authorized and unauthorized genetically modified plants. Anal Bioanal Chem 396:2065–2072

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The author thanks YeBT (Seoul, Korea) for technical support for the liquid bead array. This research was supported by the Biogreen 21 project (201003010612150010400) from the RDA, Korea.

Author information

Authors and Affiliations

  1. Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women’s University, 621 Hwarangro, Nowon, Seoul, 139-774, South Korea

    Sun Hee Choi

Authors
  1. Sun Hee Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sun Hee Choi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Choi, S.H. 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, 647–655 (2011). https://doi.org/10.1007/s00216-011-5132-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-011-5132-y

Keywords

Search

Navigation