Abstract
DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
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(3):347–354
Regulation (EC)N. 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. Edited by Commun. OJE; 2003: 1-23
Terry CF, Shanahan DJ, Ballam LD, Harris N, McDowell DG, Parkes HC (2002) Real-time detection of genetically modified soya using Lightcycler and ABI 7700 platforms with TaqMan, Scorpion, and SYBR Green I chemistries. J AOAC Int 85(4):938–944
Moreano F, Busch U, Engel KH (2005) Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation. J Agric Food Chem 53(26):9971–9979
Gryson N, Dewettinck K, Messens K (2007) Influence of cocoa components on the PCR detection of soy lecithin DNA. Eur Food Res Technol 226:247–254
Cankar K, Stebih D, Dreo T, Zel J, Gruden K (2006) Critical points of DNA quantification by real-time PCR–effects of DNA extraction method and sample matrix on quantification of genetically modified organisms. BMC Biotechnol 6:37
Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kuhl U, Lehmkuhl H, Hetzer R et al (2008) Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 9:3
Höhne M, Santisi CR, Meyer R (2002) Real-time multiplex PCR: An accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food. Eur Food Res Technol 215:59–64
Gaudron T, Peters C, Boland E, Steinmetz A, Moris G (2009) Development of a quadruplex-real-time-PCR for screening food for genetically modified organism. Eur Food Res Technol 229:295–305
ISO/IEC 17025 (2005) General requirements for the competence of testing and calibration laboratories. Edited by ISO
ISO 21571 (2005) Foodstuffs–Methods of analysis for the detection of genetically modified organisms and derived products–Nucleic acid extraction. Edited by ISO
ISO 21570 (2005) Foodstuffs–Methods of analysis for the detection of genetically modified organisms and derived products–Quantitative nucleic acid based methods. Edited by ISO
Di Bernardo G, Del Gaudio S, Galderisi U, Cascino A, Cipollaro M (2007) Comparative evaluation of different DNA extraction procedures from food samples. Biotechnol Prog 23(2):297–301
Peano C, Samson MC, Calmieri L, Gulli M, Marmiroli N (2004) Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods. J Agric Food Chem 52:6962–6968
Monma K, Araki R, Ichikawa H, Sato M, Uno N, Sato K, Tobe T, Kuribara H, Matsuoka T, Hino A, Saito K (2004) Detection of genetically modified organisms obtained from food samples. J Food Hyg Soc Japan 45(4):184–190
Acknowledgments
This study is dedicated to the memory of Prof. Antonino Cascino, who passed away on April 6, 2008. We are greatly indebted to Mrs. M.R. Cipollaro and Mrs O. Matarazzo for their administrative assistance, to Dr. Sebastiano De Biase (Merigen, Naples, Italy) for providing us with leu tRNA and NOS terminator primer and to Dr. Amalia Forte for valuable suggestions. This research was supported by "Fondo Integrativo Speciale per la Ricerca (FISR),"Tracciabilità genetica per la qualità e sicurezza della filiera della carne e quantizzazione di Organismi Geneticamente Modificati (2005–2009) to M.C. and by "Ministero delle Politiche Agricole e Forestali (MIPAF)," Progetto Speciale MIPAF: Tipizzazione e caratterizzazione di varietà precoci di patata mediante l'impiego di tecniche molecolari e spettroscopiche (2008–2011) to M.C.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Del Gaudio, S., Cirillo, A., Di Bernardo, G. et al. A preamplification approach to GMO detection in processed foods. Anal Bioanal Chem 396, 2135–2142 (2010). https://doi.org/10.1007/s00216-009-3199-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-009-3199-5