Analytical and Bioanalytical Chemistry

, Volume 396, Issue 6, pp 2023–2029 | Cite as

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

  • Meti Buh GašparičEmail author
  • Torstein Tengs
  • Jose Luis La Paz
  • Arne Holst-Jensen
  • Maria Pla
  • Teresa Esteve
  • Jana Žel
  • Kristina Gruden


Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.


Real-time PCR GMO detection TaqMan SYBR green Molecular beacons Alternative chemistries 



This study was financially supported by the European Commission through the Integrated Project Co-Extra, Contract No. 007158, under the 6th framework programme, priority 5, food quality and safety and Slovenian research agency programme Plant biotechnology and systems biology P4-0165. For help with graphical illustrations we kindly thank Damijan Gašparič, MArch.

Supplementary material

216_2009_3418_MOESM1_ESM.pdf (515 kb)
ESM 1 (PDF 515 kb)


  1. 1.
    Gruere GP, Rao SR (2007) AgBioForum 10:51–64Google Scholar
  2. 2.
    James C (2009) Global status of commercialized biotech/GM crops 2008. ISAAA brief 39, 1–20. Ithaca, NYGoogle Scholar
  3. 3.
    Holst-Jensen A, Ronning SB, Lovseth A, Berdal KG (2003) Analytical and Bioanalytical Chemistry 375:985–993Google Scholar
  4. 4.
    Holst-Jensen A (2007) Sampling, detection, identification and quantification of genetically modified organisms (GMOs). In: Food Toxicants Analysis. Techniques, Strategies and Developments. Elsevier, Amsterdam, NetherlandsGoogle Scholar
  5. 5.
    Gene Quantification []. 24-7-2009
  6. 6.
    Bustin SA (2005) Expert Rev Mol Diagn 5:493–498CrossRefGoogle Scholar
  7. 7.
    Hernandez M, Esteve T, Prat S, Pla M (2004) J Cereal Sci 39:99–107CrossRefGoogle Scholar
  8. 8.
    Terry CF, Shanahan DJ, Ballam LD, Harris N, McDowell DG, Parkes HC (2002) J AOAC Int 85:938–944Google Scholar
  9. 9.
    Andersen CB, Holst-Jensen A, Berdal KG, Thorstensen T, Tengs T (2006) J Agric Food Chem 54:9658–9663CrossRefGoogle Scholar
  10. 10.
    Buh GM, Cankar K, Zel J, Gruden K (2008) BMC Biotechnol 8:26CrossRefGoogle Scholar
  11. 11.
    La Paz JL, Esteve T, Pla M (2007) J Agric Food Chem 55:4312–4318CrossRefGoogle Scholar
  12. 12.
    Higuchi R, Fockler C, Dollinger G (1993) Watson R. Biotechnology (NY) 11:1026–1030CrossRefGoogle Scholar
  13. 13.
    Schneeberger C, Speiser P, Kury F, Zeillinger R (1995) PCR Methods Appl 4:234–238Google Scholar
  14. 14.
    Ririe KM, Rasmussen RP, Wittwer CT (1997) Anal Biochem 245:154–160CrossRefGoogle Scholar
  15. 15.
    Giglio S, Monis PT, Saint CP (2003) Nucleic Acids Res 31:e136CrossRefGoogle Scholar
  16. 16.
    Karsai A, Muller S, Platz S, Hauser MT (2002) Biotechniques 32:790–796Google Scholar
  17. 17.
    Zipper H, Brunner H, Bernhagen J, Vitzthum F (2004) Nucleic Acids Res 32:e103CrossRefGoogle Scholar
  18. 18.
    Nazarenko I, Pires R, Lowe B, Obaidy M, Rashtchian A (2002) Nucleic Acids Res 30:2089–2195CrossRefGoogle Scholar
  19. 19.
    Sherrill CB, Marshall DJ, Moser MJ, Larsen CA, Daude-Snow L, Jurczyk S, Shapiro G, Prudent JR (2004) J Am Chem Soc 126:4550–4556CrossRefGoogle Scholar
  20. 20.
    Nazarenko IA, Bhatnagar SK, Hohman RJ (1997) Nucleic Acids Res 25:2516–2521CrossRefGoogle Scholar
  21. 21.
    Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Proc Natl Acad Sci USA 88:7276–7280CrossRefGoogle Scholar
  22. 22.
    Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J (2000) Nucleic Acids Res 28:655–661CrossRefGoogle Scholar
  23. 23.
    Costa JM, Ernault P, Olivi M, Gaillon T, Arar K (2004) Clin Biochem 37:930–932CrossRefGoogle Scholar
  24. 24.
    Koshkin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, Olsen CE, Wengel J (1998) Tetrahedron 54:3607–3630CrossRefGoogle Scholar
  25. 25.
    Duck P, Varado-Urbina G, Burdick B, Collier B (1990) Biotechniques 9:142–148Google Scholar
  26. 26.
    Tyagi S, Kramer FR (1996) Nat Biotechnol 14:303–308CrossRefGoogle Scholar
  27. 27.
    European Network of GMO Laboratories (ENGL). Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing (2008). 2009
  28. 28.
    Santangelo P, Nitin N, Bao G (2006) Ann Biomed Eng 34:39–50CrossRefGoogle Scholar
  29. 29.
    Mao F, Leung WY, Xin X (2007) BMC Biotechnol 7:76CrossRefGoogle Scholar
  30. 30.
    Monis PT, Giglio S, Saint CP (2005) Anal Biochem 340:24–34CrossRefGoogle Scholar
  31. 31.
    AlleLogic Biosciences Corp []. 1-8-2009
  32. 32.
    Christensen UB (2008) Methods Mol Biol 429:137–160CrossRefGoogle Scholar
  33. 33.
    ISO 21569:2005. Foodstuffs—Methods of analysis for the detection of genetically modified organisms and derived products—Qualitative nucleic acid based methods. 1–69. 20-6-2005. Geneva, Switzerland; 2005Google Scholar
  34. 34.
    Pfaffl MW (2001) Nucleic Acids Res 29:e45CrossRefGoogle Scholar
  35. 35.
    ISO 24276:2006. Foodstuffs—Methods of analysis for the detection of genetically modified organisms and derived products—General requirements and definitions. 1–16. 20-6-2005. Geneva, Switzerland; 2005Google Scholar
  36. 36.
    Berdal KG, Holst-Jensen A (2001) Eur Food Res Technol 213:432–438CrossRefGoogle Scholar
  37. 37.
    Hernandez M, Pla M, Esteve T, Prat S, Puigdomenech P, Ferrando A (2003) Transgenic Res 12:179–189CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Meti Buh Gašparič
    • 1
    Email author
  • Torstein Tengs
    • 2
  • Jose Luis La Paz
    • 3
  • Arne Holst-Jensen
    • 2
  • Maria Pla
    • 3
  • Teresa Esteve
    • 3
  • Jana Žel
    • 1
  • Kristina Gruden
    • 1
  1. 1.Department of Biotechnology and Systems BiologyNational Institute of BiologyLjubljanaSlovenia
  2. 2.Section for food bacteriology and GMONational Veterinary InstituteOsloNorway
  3. 3.Consorci CSIC-IRTA and IBMB-CSICBarcelonaSpain

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