Biotechnologie, Agronomie, Société et Environnement (Jan 2018)
Development of PCR screening assays focused on gene-coding sequences for GMO detection
Abstract
Description of the subject. This paper describes the development, evaluation and limitations of screening PCR assays based on genes and for use in GMO detection. Objectives. The aim of this research is to propose new PCR assays based on gene-coding sequences that will join a panel of existing screening assays to make better GMO detection possible. Method. Real-time PCR methods using double-dye probes were developed for genes frequently encountered in GM constructs and evaluated in terms of specificity and sensitivity. Results. Eight real-time PCR tests were designed based on the sequences of the bar, pat, EPSPS, gox, gus and hsp70 genes. Two of them proved of limited interest due to the presence of positive signals linked to the presence of the donor organisms: residual sequences of Escherichia coli in the master mixes for the gus PCR assay and while for the hsp70 PCR test, hsp70 being only used in GM maize at present, it is useless as hsp70 originates from maize. The assays for bar, pat, EPSPS and gox were found to successfully detect the corresponding structural elements introduced in GM constructs. Several PCR assays were proposed for the EPSPS gene in order to cover the different versions of the gene. Conclusions. Real-time PCR tests for bar, pat, EPSPS and gox were developed and met the expected performance criteria in terms of specificity and sensitivity. The targets can be amplified with the same PCR conditions as PCR assays already developed for the detection of promoters and terminators, and can be used in combination on the same PCR plate in order to provide wider coverage of GMOs and initial information concerning the GMO(s) present.
