Skip to main content
SpringerLink
Log in

A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials

  • Original Paper
  • Published:
European Food Research and Technology Aims and scope Submit manuscript

Abstract

This paper is the first part of a serial study investigating a quantification model freed from endogenous reference gene for genetically modified (GM) content by real-time polymerase chain reaction (PCR). The serial study involves two parts: (1) quantitative determination of GM DNA in haplo-species plant samples; (2) quantitative determination of GM DNA in multi-species plant samples. The paper describes a methodology to quantify the GM content in a DNA extract using on one hand real-time PCR to determine the amount of GM targets present and on the other hand absorbance reading at 260 nm to measure the total DNA present in sample. The ratio of both values is expressed as GM percentage. The most prominent dominance of the novel model is that the direct quantitative relation between the initial amount of target template in real-time PCR reaction (X 0) and the content of GM DNA in tested material (X SD) is established. Theoretical analysis indicates that the developed quantitative model relieved from the dependence on endogenous reference genes is suitable to quantify the GM content in haplo-species plant sample, in addition, it has the applicability in the quantitative detection of GM content in multi-species GM plant sample. A trail, in which 75 haplo-species GM plant samples were involved, was conducted to validate the suitability of the novel quantification model. The bias varied from 0.00 to 24.00% except a tested sample with lower level of GM content, and the precision expressed as coefficient of variation (CV) was from 2.81 to 25.00%. The limit of quantitation (LOQ) of the quantitative assay was as low as 0.1%. Compared with the previous papers and the performance requirements raised by European Network of GMO Laboratories (ENGL) for analytical methods of GMO testing, the results demonstrated that the established quantification model is a suitable alternative to the more traditional endogenous reference assay in the quantification of GM content in haplo-species plant sample.

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

Similar content being viewed by others

Abbreviations

C D :

The DNA content in tested materials

CE:

Capillary electrophoresis

C t :

Cycle threshold

CRMs:

Certified reference materials

CV:

The coefficient of variation

DNA:

Deoxyribonucleic acid

dsDNA:

Double-strands DNA

e :

The base number of natural logarithm

E :

Efficiency of the PCR reaction

E ex :

DNA extraction efficiency

ELISA:

Enzyme linked immunosorbent assay

ENGL:

European Network of GMO Laboratories

EU:

European Union

FAM:

6-Carboxy-fluorescein

GC:

Gas chromatography

GM:

Genetically modified

GMO:

Genetically modified organism

HPLC:

High performance liquid chromatography

K :

A constant

LOQ:

Limit of quantitation

M g :

Genome molecular weight

M gR :

The genome molecular weight of the species that GMO belongs to

M gX :

The genomic molecular weight of GMO

N C :

The copy number of target exogenous gene in GMO genome

N CR :

The copy number of endogenous reference gene in genome

N CX :

The copy number of exogenous target gene in genome

OD260 :

Optical density at 260 nm

OD320 :

Optical density at 320 nm

p35S:

35S Promoter from Cauliflower Mosaic Virus

PCR:

Polymerase chain reaction

R 0 :

The initial amount of endogenous gene in PCR reaction

R 2 :

Correlation coefficient

R D :

The DNA content of total species

RRS:

Roundup Ready™ soybean line GT 40-3-2

R S :

The mass ratio of total ingredients of a certain species plant to the total ingredients of the tested materials

RSD:

Relative standard deviation

SD:

Standard deviation

tNOS:

Terminator of nopaline synthase gene

TAMRA:

6-Carbixytetramethylrhodamine

V 1 :

The total volume of DNA extraction solution of tested materials

V 2 :

The volume of DNA solution added in PCR reaction

W :

The mass of tested materials

X 0 :

The initial amount of target template in PCR reaction

W T-DNA :

The mass of GM DNA in the serial PCR reaction

W S-DNA :

The DNA mass of tested materials in PCR reaction

X D :

The DNA content in GMO

X S :

The mass ratio of total ingredients of GMO to the total ingredients of the tested materials

X SD :

The mass% of GM DNA to the total DNA in tested materials

References

  1. Hardegger M, Brodmann P, Herrmann A (1999) Quantitative detection of the 35S promoter and NOS terminator using quantitative competitive PCR. Eur Food Res Technol 209:83–87

    Article  Google Scholar 

  2. Regulation (EC) Number 1830/2003 (2003) concerning the traceability and labeling of genetically modified organisms and the traceability of food and feed products produced from GMOs. Off J Eur Union 268:24–28

    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  Google Scholar 

  4. Huang CC, Pan TM (2005) Event-specific real-time detection and quantification of genetically modified Roundup Ready soybean. J Agric Food Chem 53:3833–3839

    Article  Google Scholar 

  5. Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H, Jan H (2006) Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression′s CT difference” formula. J Mol Med 84:901–910

    Article  Google Scholar 

  6. Weng HB, Pan AH, Yang LT, Zhang CM, Liu ZL, Zhang DB (2004) Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Mol Biol Rep 22:289–300

    Article  Google Scholar 

  7. Gulden RH, Lerat S, Hart MM, Powell JR, Trevors JT, Pauls KP, Klironomos JN, Swanton CJ (2005) Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis. J Agric Food Chem 53(15):5858–5865

    Article  Google Scholar 

  8. Metzdorff SB, Kok EJ, Knuthsen P, Pedersen J (2006) Evaluation of a non-targeted “Omic” approach in the safety assessment of genetically modified plants. Plant Biol 8:662–672

    Article  CAS  Google Scholar 

  9. Varzakas TH, Chryssochoidis G, Argyropoulos D (2007) Approaches in the risk assessment of genetically modified foods by the Hellenic Food Safety Authority. Food Chem Toxicol 45:530–542

    Article  Google Scholar 

  10. Miraglia M, Berdal KG, Brera C, Corbisier P, Holst-Jensen A, Kok EJ, Marvin HJP, Schimmel H, Rentsch J, Rie JPPF, van Zagon J (2004) Detection and traceability of genetically modified organisms in the food production chain. Food Chem Toxicol 42:1157–1180

    Article  CAS  Google Scholar 

  11. European Commission Recommendation 2004/787/EC (2004) On technical guidance for sampling and detection of genetically modified organisms and materials produced from genetically modified organisms as or in products in the context of Regulation (EC) No. 1830/2003. Off J Eur Union L 348:18–26

  12. Burns M, Corbisier P, Wiseman G, Valdivia H, McDonald P, Bowler P, Ohara K, Schimmel H, Charels D, Damant A, Harris N (2006) Comparison of plasmid and genomic DNA calibrants for the quantification of genetically modified ingredients. Eur Food Res Technol 224:249–258

    Article  Google Scholar 

  13. Roda A, Mirasoli M, Guardigli M, Michelini E, Simoni P, Magliulo M (2006) Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize. Anal Bioanal Chem 384:1269–1275

    Article  CAS  Google Scholar 

  14. Van den Bulcke M, De Schrijver A, De Bernardi D, Devos Y, MbongoMbella G., Leunda Casi A, Moens W, Sneyers M (2007) Detection of genetically modified plant products by protein strip testing: an evaluation of real-life samples. Eur Food Res Technol 225:49–57

    Article  Google Scholar 

  15. Annette B, Gerhard S (2003) Validation of different genomic and cloned DNA calibration standards for construct-specific quantification of LibertyLink in rapeseed by real-time PCR. Eur Food Res Technol 216:421–427

    Google Scholar 

  16. European Commission, Joint Research Centre, Institute for Health and Consumer Protection (2001) Review of GMO detection and quantification techniques

  17. Moreano F, Busch U, Engel KH (2005) Distortion of genetically modified organism quantification in processed foods: influence of particel size compositions and heat-induced DNA degradation. J Agric Food Chem 53:9971–9979

    Article  Google Scholar 

  18. Engel KH, Moreano F, Ehlert A, Busch U (2006) Quantification of DNA from genetically modified organisms in composite and processed foods. Trends Food Sci Technol 17:490–497

    Article  Google Scholar 

  19. James C (2005) Executive summary of global status of commercialized biotech/GM crops. ISAAA Briefs No 34

  20. Wilhelm J, Pingoud A, Hahn M (2003) Real-time PCR-based method for the estimation of genome sizes. Nucleic Acids Res 31(10):e56

    Article  Google Scholar 

  21. Inspection and Quarantine Trade of China (2003) Inspection and Quarantine Trade Standard of China. Protocol of the real-time PCR for detecting genetically modified plants and their derived products

  22. Rott ME, Lawrence TS, Wall EM, Green MJ (2004) Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction. J Agric Food Chem 52:5223–5232

    Article  Google Scholar 

  23. Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N, Hirao T, Akiyama H, Goda Y, Toyoda M, Hino A (2002) Novel reference molecules for quantitation of genetically modified maize and soybean. J AOAC Int 85:1077–1189

    Google Scholar 

  24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2­ΔΔCt method. Methods 25(4):402–408

    Article  CAS  Google Scholar 

  25. Deng PJ, Yang DY, Li BL, Yang YC (2005) The theory study on mathematical equation of GMIs quantitative analysis by real-time fluorescence PCR. China J Health Lab Technol 15(7):769–772

    Google Scholar 

  26. Yang DY, Deng PJ, Li BL, Yang YC (2005) The study of validation on mathematical equation of GMIs quantitative analysis by Real-time fluorescence PCR. South China J Prev Med 31(Suppl):556–560

    Google Scholar 

  27. Wang GL, Fang HJ (2002) Plant Genetic Engineering. Science Publishing House, Beijing, China. 2002, 755

  28. Weng HB, Yang LT, Liu ZL, Ding JY, Pan AH, Zhang DB (2005) Novel reference gene, high-mobility-group protein I/Y, used in qualitative and real-time quantitative polymerase chain reaction detection of transgenic rapeseed cultivars. J AOAC Int 88:577–584

    Google Scholar 

  29. Yang L, Chen JX, Huang C, Liu Y, Jia S, Pan LW, Zhang DB (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  Google Scholar 

  30. Charels D, Broeders S, Corbisier P, Trapmann S, Schimmel H, Linsinger T, Emons H (2007) Toward metrological traceability for DNA fragment ratios in GM quantification. 2. Systematic study of parameters influencing the quantitative determination of MON 810 corn by real-time PCR. J Agric Food Chem 55:3258–3267

    Article  Google Scholar 

  31. European Network of GMO Laboratories (ENGL) (25/01/2005) Definition of minimum performance requirements for analytical methods of GMO testing

  32. Codex Alimentarius Commission, ALINORM 97/23A, Appendix 3

Download references

Acknowledgments

This work was supported by Science and Technology Fundation of Guangdong Province (NO.2005B26001116); Natural Science Fundation of Guangdong Province (NO.06027682); and Shenzhen Bereau of Science Tecnology and Information [(2007) 334–339].

Author information

Authors and Affiliations

  1. Shenzhen Center for Disease Control and Prevention, No. 21, 1st Road of Tianbei, 518020, Shenzhen, People’s Republic of China

    Pingjian Deng, Dongyan Yang, Yongcun Yang & Xiaoke Yang

  2. College of Animal Science, South China Agricultural University, Wushan Road, 510642, Guangzhou, People’s Republic of China

    Liangrang Guo

  3. Center for Quality Crop Seed of ShenZhen Municipality, F3, 213 Building, 9th Tairan Road of Chegongmiao, 518040, Shenzhen, People’s Republic of China

    Xiangyang Zhou & Xueling Wang

Authors
  1. Pingjian Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dongyan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yongcun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaoke Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Liangrang Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiangyang Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xueling Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pingjian Deng.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Deng, P., Yang, D., Yang, Y. et al. A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials. Eur Food Res Technol 227, 1485–1498 (2008). https://doi.org/10.1007/s00217-008-0871-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00217-008-0871-5

Keywords

Search

Navigation