Four new SYBRGreen qPCR screening methods for the detection of Roundup Ready, LibertyLink, and CryIAb traits in genetically modified products

摘要

SYBR®Green qPCR methods for the detection of the Roundup Ready® Cp4-EPSPS, LibertyLink® “PAT” and“BAR” and the Bacillus thuringiensis CryIAb/Ac traits as present in genetically modified organisms (GMO)were developed. The specificity, sensitivity, dynamic range and PCR method efficiency was determined for eachof them. All methods comply with the performance criteria set by the European Network of GMO Laboratories(ENGL), ISO norms and Codex guidelines. The developed methods are specific and generate amplicons of 108,73, 109 and 69 bp respectively for “Cp4-EPSPS”, “CryIAb/Ac”, “PAT” and “BAR” targets. Single-targetSybricon plasmids comprising the corresponding PCR amplicons have been constructed and used as controls inPCR analysis. These four SYBR®Green qPCR methods for GMO analysis complete the set of qPCR methodsapplied in the so-called combinatory SYBR®Green qPCR screening (abbreviated as "CoSYPS''; Van den Bulckeet al, 2010, Anal Bioanal Chem, 396(6):2113-23). Due to their trait-specific nature, these methods allowdiscriminating better between the different GMO. Compared to GMO screening applying only common targetssuch as the 35S promoter or the NOS terminator, screening with trait-specific methods reduces the number ofpossible GMO candidates present in a sample, reducing the cost of the analysis. The application of thesemethods in CoSYPS analysis is demonstrated using two GEMMA proficiency test samples and referencematerial from the genetically modified rapeseed event GT73. This set of SYBR®Green qPCR trait-specificmethods represents a very interesting novel set of GMO analysis methods allowing cost-effective identificationof GM materials in products.