Abstract
Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers’ perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.
Abbreviations
- CRM:
-
Certified reference material
- GMO:
-
Genetically modified organisms
- IPC:
-
Internal positive control
- LOD:
-
Limit of detection
- MGB:
-
Minor groove binder
- NTC:
-
No template control
- PCR:
-
Polymerase chain reaction
- RTi-PCR:
-
Real-time PCR
References
James C (2011) Global status of commercialized biotech/GM crops: 2011. ISAAA Briefs 2011:43
Stein AJ, Rodriguez-Cerezo E (2009) The global pipeline of new GM crops: implications of asynchronous approval for international trade. JRC Sci Tech Rep. doi:10.2791/12087
Ruane J (2006) An FAO e-mail conference on GMOs in the pipeline in developing countries: the moderator’s summary. FAO. http://www.fao.org/biotech/biotech-forum/. Accessed 23 January 2013
Ruebelt MC, Lipp M, Reynolds TL, Astwood JD, Engel KH, Jany KD (2006) Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 2. Assessing analytical validation. J Agric Food Chem 54:2162–2168
Kim Y-H, Choi SJ, Lee H-A, Moon TW (2006) Quantitation of CP4 5-enolpyruvylshikimate-3-phosphate synthase in soybean by two-dimensional gel electrophoresis. J Microbiol Biotechn 6:25–31
Latoszek A, García-Ruiz C, Marina ML, De La Mata FJ, Gómez R, Rasines B, Cifuentes A, Pobozy E, Trojanowicz M (2011) Modification of resolution in capillary electrophoresis for protein profiling in identification of genetic modification in foods. Croat Chem Acta 84:375–382
López MCG, Garcia-Cañas V, Alegre MLM (2009) Reversed-phase high-performance liquid chromatography-electrospray mass spectrometry profiling of transgenic and non-transgenic maize for cultivar characterization. J Chromatogr A 1216:7222–7228
Xu W, Huang K, Liang Z, Deng A, Yuan Y, Guo F, Luo Y (2009) Application of stepwise ammonium sulfate precipitation as cleanup tool for an enzyme-linked immunosorbent assay of glyphosate oxidoreductase in genetically modified rape of gt73. J Food Biochem 33:630–648
Elenis DS, 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 392:347–354
Marmiroli N, Maestri E, Gullì M, Malcevschi A, Peano C, Bordoni R, De Bellis G (2008) Methods for detection of GMOs in food and feed. Anal Bioanal Chem 392:369–384
International Organization for Standardization (2005) ISO 21569:2005. Foodstuffs—methods of analysis for the detection of genetically modified organisms and derived products—qualitative nucleic acid based methods. International Organization for Standardization, Geneva, Switzerland
nternational Organization for Standardization (2005) ISO 21570:2005. Foodstuffs—methods of analysis for the detection of genetically modified organisms and derived products—quantitative nucleic acid based methods. International Organization for Standardization, Geneva, Switzerland
Dörries H-H, Remus I, Grönewald A, Grönewald C, Berghof-Jäger K (2010) Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs). Anal Bioanal Chem 396:2043–2054
Guo J, Chen L, Liu X, Gao Y, Zhang D, Yang L (2012) A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs. Food Chem 132:1566–1573
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 Method 2:325–336
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) 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 28:655–661
Gasparic MB, Tengs T, La Paz JL, Holst-Jensen A, Pla M, Esteve T, Zel J, Gruden K (2010) Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Anal Bioanal Chem 396:2023–2029
International Organization for Standardization (2006) ISO 24276:2006. Foodstuffs—methods of analysis for the detection of genetically modified organisms and derived products—general requirements and definitions. International Organization for Standardization, Geneva, Switzerland
European Commission (2011) EC decision (2011/884/EU) on emergency measures regarding unauthorised genetically modified rice in rice products originating from China and repealing Decision 2008/289/EC. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2011:343:0140:0148:EN:PDF. Accessed 28 March 2012
Ghedira R, Papazov N, Vuylsteke M, Ruttink T, Taverniers I, De Loose M (2009) Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification. J Agric Food Chem 57:9370–9377
Gryson N (2010) Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Anal Bioanal Chem 396:2003–2022
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Arumuganathan K, Earle ED (1991) Nuclear content of some important plant species. Plant Mol Biol Rep 9:208–218
Joint Research Center, European Union Reference Laboratory for GM Food & Feed (EURL), Ispra, Italy (2011) Technical guidance document from the European Union Reference Laboratory for genetically modified food and feed on the implementation of commission regulation (EU) no 619/2011. http://gmo-crl.jrc.ec.europa.eu/doc/Technical%20Guidance%20from%20EURL%20on%20LLP.pdf. Accessed 14 May 2013
Wu G, Zhang L, Wu Y, Cao Y, Lu C (2010) Comparison of five endogenous reference genes for specific PCR detection and quantification of Brassica napus. J Agric Food Chem 58:2812–2817
Schmidt A-M, Rott ME (2006) Real-time polymerase chain reaction (PCR) quantitative detection of Brassica napus using a locked nucleic acid TaqMan probe. J Agric Food Chem 54:1158–1165
CERA (2013) CERA GM crop database, ILSI Research Foundation, Washington DC, USA. http://cera-gmc.org/index.php?action=gm_crop_database&mode=ShowProd&data=176. Accessed 28 March 2012
Institute for Reference Materials and Measurements (IRMM), Geel, Belgium (2007) Additional information related to the raw material used by IRMM for the production of GMO CRMs. http://irmm.jrc.ec.europa.eu/reference_materials_catalogue/user_support/Documents/FAQ%20GMO_attachment.pdf. Accessed 24 April 2012
Joint Research Center, European Union Reference Laboratory for GM Food & Feed (EURL), Ispra, Italy (2008) Definition of minimum performance requirements for analytical methods of GMO testing. http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm. Accessed 19 December 2012
Laube I, Hird H, Brodmann P, Ullmann S, Schöne-Michling M, Chisholm J, Broll H (2010) Development of primer and probe sets for the detection of plant species in honey. Food Chem 118:979–986
Bahrdt C, Krech AB, Wurz A, Wulff D (2010) Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed. Anal Bioanal Chem 396:2103–2112
Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L (2004) Polyphenols: food sources and bioavailability. Am J Clin Nutr 79:727–747
Demeke T, Jenkins GR (2010) Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem 396:1977–1990
Chaouachi M, El Malki R, Berard A, Romaniuk M, Laval V, Brunel D, Bertheau Y (2008) Development of a real-time PCR method for the differential detection and quantification of four Solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melogena), and pepper (Capsicum anuum). J Agric Food Chem 56:1818–1828
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 PCR quantitative PCR detection of transgenic cottons. Plant Cell Rep 24:237–245
Collonier C, Schattner A, Berthier G, Boyer F, Coué-Philippe G, Diolez A, Duplan MN, Fernandez S, Kebdani N, Kobilinski A, Romaniuk M, De Beuckeleer M, De Loose M, Windels P, Bertheau Y (2005) Characterization and event specific-detection by quantitative real-time PCR of T25 maize insert. J AOAC Int 88:536–546
Takabatake R, Akiyama H, Sakata K, Onishi M, Koiwa T, Futo S, Minegishi Y, Teshima R, Mano J, Furui S, Kitta K (2011) Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12. Food Hyg Saf Sci 52:100–107
Tengs T, Kristoffersen AB, Zhang H, Berdal KG, Lovoll M, Holst-Jensen A (2010) Non-prejudiced detection and characterization of genetic modifications. Food Anal Methods 3:120–128
Mäde D, Degner C, Grohmann L (2006) Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice. Eur Food Res Technol 224:271–278
Acknowledgments
We would like to thank Frederic Aymes, Celeste Chia, Hui Zhen Ho and Yuying Zhong from Nestlé Quality Assurance Center, Singapore, for their valuable contribution, as well as Matthias Kiehne from Life Technologies for his support on this project.
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Cottenet, G., Blancpain, C., Sonnard, V. et al. Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms. Anal Bioanal Chem 405, 6831–6844 (2013). https://doi.org/10.1007/s00216-013-7125-5
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DOI: https://doi.org/10.1007/s00216-013-7125-5