Untitled

Abstract

The increasing presence of transgenic plant derivatives in a wide range of animal and human consumables has provoked in western Europe a strong demand for appropriate detection methods to evaluate the existence of transgenic elements. Among the different techniques currently used, the real-time quantitative PCR is a powerful technology well adapted to the mandatory labeling requirements in the European Union (EU). The use of transgene flanking genomic sequences has recently been suggested as a means to avoid ambiguous results both in qualitative and quantitative PCR-based technologies. In this study we report the identification of genomic sequences adjacent to the 3'-integration site of event MON810 in transgenic maize. This genetically modified crop contains transgene sequences leading to ectopic expression of a synthetic CryIA(b) endotoxin which confers resistance to lepidopteran insects especially against the European corn borer. The characterization of the genome-transgene junction sequences by means of TAIL-PCR has facilitated the design of a specific, sensitive and accurate quantification method based on TaqMan chemistry. Cloning of event MON810 3'-junction region has also allowed to compare the suitability of plasmid target sequences versus genomic DNA obtained from certified reference materials (CRMs), to prepare standard calibration curves for quantification.

Quantitative Detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly , iap , and lin02483 Targets and AmpliFluor Technology

“…The RTi-PCR assays that we developed could detect approximately one target genome in at least 11% of the experiments, 3 CFU in 55 to 89% of replicates, and 30 CFU in all cases. Detection limits were similar to those previously reported for other RTi-PCR assays targeting single-copy genes (14,15,16,29). Limits of detection of 9 (18), 6 to 60 (29), and 500 (24) target molecules were previously reported for hly, and six genome copies were reported for iap (12).…”

Assessment of Genetically Modified Organisms (GMO) in Meat Products by PCR

“…Different strategies based on PCR amplifi cation of the introduced transgene have been defi ned : traitspecifi c methods, where transgene -specifi c sequence are used for amplifi cation (Va ï tilingom et al 1999 ;Zeitler et al 2002 ); construct or plasmid -specifi c methods, with the amplifi cation of a fragment containing two introduced regions from different origins (Kuribara et al 2002 ;Hernandez et al 2004a ); and event -specifi c methods, which amplify the border region between the introduced sequence and the plant genomic DNA (e.g., for Roundup Ready soybean: Berdal and Holst -Jensen 2001 ;Taverniers et al 2001 ;Terry and Harris 2001 ), for transgenic maize lines, such as MON810 (Holck et al 2002 ;Hernandez et al 2003a ), CBH -351 (Windels et al 2003 ), Bt11 (Zimmermann et al 2000 ), and NK603 (Nielsen et al 2004 ). (Cankar et al 2005 ).…”

“…Consequently, the ideal DNA target sequence for GMO quantifi cation is a sequence found only once in a stable and known copy number per genome. For this reason, several works describe the characterization of junction regions between the host plant genome and the transgene (Zimmermann et al 2000 ;Holck et al 2002 ;Hernandez et al 2003a ;Windels et al 2003 ;Nielsen et al 2004 ;Taverniers et al 2004 ) that are unique for a single transformation event (event -specifi c). Only one copy of …”

“…While the genomic certifi ed material better mimics the target of detection in the real sample, it is sometimes diffi cult to obtain and is expensive. The use of plasmids as standards has been recently introduced (Hernandez et al 2003a ;Taverniers et al 2004Taverniers et al , 2005Toyota et al 2006 ), with the advantages of being Schmittgen 2001 ); recently, for example, a validated quantitative RTi -PCR method for MON 810 has been assessed using this approach (Aguilera et al 2009 ). The lowest serial dilution allows the determination of absolute detection and quantifi cation limits of the method, while a correct determination of the practical quantifi cation limits requires a thorough statistical examination of the sampling procedure when preparing the serial dilutions, based on binomial distributions and Monte -Carlo simulations (Hernandez et al 2003a ).…”

“…The use of plasmids as standards has been recently introduced (Hernandez et al 2003a ;Taverniers et al 2004Taverniers et al , 2005Toyota et al 2006 ), with the advantages of being Schmittgen 2001 ); recently, for example, a validated quantitative RTi -PCR method for MON 810 has been assessed using this approach (Aguilera et al 2009 ). The lowest serial dilution allows the determination of absolute detection and quantifi cation limits of the method, while a correct determination of the practical quantifi cation limits requires a thorough statistical examination of the sampling procedure when preparing the serial dilutions, based on binomial distributions and Monte -Carlo simulations (Hernandez et al 2003a ). Finally, careful analysis and interpretation of data from the real -time PCR method have to be performed properly, with the use of the correct calculation and interpretation of correlation coeffi cients and regression techniques, especially when traces are analyzed (Burns et al 2004 ).…”