European Food Research and Technology

, Volume 230, Issue 2, pp 239–248 | Cite as

Development and in-house validation of a reference molecule pMIR604 for simplex and duplex event-specific identification and quantification of GM maize MIR604

Original Paper

Abstract

Effective analysis methods for genetically modified organisms (GMOs) using reliable reference molecules as calibrators are necessary for the implementation of labeling policies. While no available detection systems based on the reference molecule have been reported for GM maize MIR604. Here we established the simplex and duplex qualitative and quantitative PCR systems for maize MIR604 employing a reliable new reference molecule pMIR604 as a calibrator, which contains fragments of the revealed 5′ flanking sequence of maize MIR604 and taxon-specific sequence zSSIIb. The limits of detection (LODs) were both 10 copies. The absolute LOD and limit of quantification were confirmed to be as low as 10 and 25 copies of pMIR604 through in-house validation both in simplex and duplex real-time PCR assays. For quantification of practical samples, results from five operators indicated that the biases of the data derived from each participant ranged from 2.00 to 23.00% and from 3.00 to 24.00% in simplex and duplex PCR systems, respectively. The relative standard deviations of the mean values for different GM content maize samples were all within 13.60% both in simplex and duplex analyses. This study showed that the detection systems of GM maize MIR604 using a new reference molecule pMIR604 as a calibrator are applicable for analysis of GM maize MIR604 and suitable for use as a preferable substitute of the reference material derived from plant raw materials.

Keywords

Duplex PCR Event specific Genetically modified organism MIR604 Reference molecule 

References

  1. 1.
    James C (2008) Global status of commercialize biotech/GM crops. ISAAA Briefs 39Google Scholar
  2. 2.
    Elenis DR, Kalogianni DP, Glynou K, Ioannou PC, Christopoulos TK (2008) Advances in molecular techniques for the detection and quantification of genetically modified organisms. Anal Bioanal Chem 392:347–354CrossRefGoogle Scholar
  3. 3.
    Holst-Jensen A, Rønning SB, Løvseth A, Berdal KG (2003) PCR technology for screening and quantification. Anal Bioanal Chem 375:985–993Google Scholar
  4. 4.
    Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N (2002) Novel reference molecules for quantitation of genetically modified maize and soybean. J AOAC Int 85:1077–1089Google Scholar
  5. 5.
    Lee SH, Min DM, Kim JK (2006) Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863. J Agric Food Chem 54:1124–1129CrossRefGoogle Scholar
  6. 6.
    Rho JK, Lee T, Jung SI, Kim TS, Park YH, Kim YM (2004) Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes. J Agric Food Chem 52:3269–3274CrossRefGoogle Scholar
  7. 7.
    Yang L, Pan A, Zhang K, Guo J, Yin C, Chen J (2005) Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods. J Agric Food Chem 53:6222–6229CrossRefGoogle Scholar
  8. 8.
    Yang L, Pan A, Zhang K, Yin C, Qian B, Chen J (2005) Qualitative and quantitative PCR methods for event-specific detection of genetically modified cotton Mon1445 and Mon531. Transgenic Res 14:817–831CrossRefGoogle Scholar
  9. 9.
    Yang L, Guo J, Pan A, Zhang H, Zhang K, Wang Z, Zhang D (2007) Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule. J Agric Food Chem 55:15–24CrossRefGoogle Scholar
  10. 10.
    Corbisier P, Broeders S, Charels D, Trapmann S, Vincent S, Emons H (2007) Certification of plasmidic DNA containing MON 810 maize DNA fragments, ERM®-AD413. European Commission, Joint Research Centre, Institute for Reference Materials and MeasurementsGoogle Scholar
  11. 11.
    Dong W, Yang L, Shen K, Kim B, Kleter GA, Marvin HJP, Guo R, Liang W, Zhang D (2008) GMDD: a database of GMO detection methods. BMC Bioinformatics 9:260–268CrossRefGoogle Scholar
  12. 12.
    Savini C, Mones W, Querci M, Mazzara M, Cordeil S, Van den Eede G, Zhang D (2007) Event-specific method for the quantification of maize line MIR604 using real-time PCR protocol. European Commission, Joint Research Centre, Institute for Health and Consumer Protection Biotechnology & GMOs UnitsGoogle Scholar
  13. 13.
    Pan A, Yang L, Xu S, Yin C, Zhang K, Wang Z, Zhang D (2006) Event-specific qualitative and quantitative PCR detection of MON863 maize based upon the 3′-transgene integration sequence. J Cereal Sci 43:250–257CrossRefGoogle Scholar
  14. 14.
    Ministry of Agriculture of People’s Republic of China (2007) Bulletin No. 953 and 869 of Ministry of AgricultureGoogle Scholar
  15. 15.
    Zhang H, Yang L, Guo J, Li X, Jiang L, Zhang D (2008) Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of Roundup Ready soybean. J Agric Food Chem 56:5514–5520CrossRefGoogle Scholar
  16. 16.
    Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218CrossRefGoogle Scholar
  17. 17.
    Trapmann S, Conneely P, Contreras M, Corbisier P, Gioria S, Van-Nyen M, Vincent S, Emonsn H (2006) The certification of reference materials of dry-mixed maize powder with different mass fractions of MIR604 maize. European Commission, Joint Research Centre, Institute for Reference Materials and MeasurementsGoogle Scholar
  18. 18.
    Zhang D, Corlet A, Fouilloux S (2007) Impact of genetic structures on haploid genomes based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications. Transgenic Res 17:393–402CrossRefGoogle Scholar
  19. 19.
    Savini C, Mones W, Querci M, Mazzara M, Cordeil S, Van den Eede G, Zhang D (2007) Event-specific method for the quantification of maize line MIR604 using real-time PCR protocol. EU-CRLGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.GMO Detection Laboratory, SJTU-Bor Luh Food Safety Center, Bio-X Research Center, Key Laboratory of Genetics & Development and Neuropsychiatric Diseases, Ministry of Education, School of life Science and BiotechnologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China
  2. 2.GMO Detection LaboratoryShanghai Entry–Exit Inspection and Quarantine BureauShanghaiPeople’s Republic of China

Personalised recommendations