Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum).
The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.
Chaouachi
Maher
M
Unité Etude du Polymorphisme des Génomes Végétaux (EPGV) UR1279, Centre National de Génotypage (CNG), 2 rue Gaston Crémieux 91057, CP5721, Evry cedex, France.
El Malki
Redouane
R
Berard
Aurélie
A
Romaniuk
Marcel
M
Laval
Valérie
V
Brunel
Dominique
D
Bertheau
Yves
Y
D001483:Base Sequence; D002212:Capsicum / Q000145:classification / Q000235:genetics*; D018744:DNA, Plant / Q000032:analysis / Q000737:chemistry; D018551:Solanum lycopersicum / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D032323:Solanum melongena / Q000145:classification / Q000235:genetics*; D011198:Solanum tuberosum / Q000145:classification / Q000235:genetics*; D043324:beta-Fructofuranosidase / Q000235:genetics
D018744:DNA, Plant; D043324:beta-Fructofuranosidase