A preamplification approach to GMO detection in processed foods
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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.
KeywordsGMO detection Real-time PCR Preamplification
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.
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