title: Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.
abstract: In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.
authors.affiliation: Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, European Commission Joint Research Centre , Via E. Fermi 2749, 21027 Ispra (VA), Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Jacchia
authors.forename: Elena
authors.initials: E
authors.lastname: Nardini
authors.forename: Christian
authors.initials: C
authors.lastname: Savini
authors.forename: Mauro
authors.initials: M
authors.lastname: Petrillo
authors.forename: Alexandre
authors.initials: A
authors.lastname: Angers-Loustau
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Shim
authors.forename: Kurniawan
authors.initials: K
authors.lastname: Trijatmiko
authors.forename: Joachim
authors.initials: J
authors.lastname: Kreysa
authors.forename: Marco
authors.initials: M
authors.lastname: Mazzara
mesh_terms: D018744:DNA, Plant / Q000032:analysis; D012275:Oryza / Q000737:chemistry*; D010739:Phospholipase D / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000145:classification* / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D014801:Vitamin A / Q000032:analysis
chemical_list: D018744:DNA, Plant; D014801:Vitamin A; D010739:Phospholipase D
keywords: Golden Rice 2 (GR2); endogenous taxon-specific assay; event-specific method; genetically modified organism (GMO); in-house validation; quantitative real-time PCR (qPCR)