Quantification of the 35S promoter in DNA extracts from genetically modified organisms using real-time polymerase chain reaction and specificity assessment on various genetically modified organisms, part I: operating procedure.
A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.
Fernandez
Sophie
S
Laboratoire Méthodologies de la Détection des OGM, Unite PMDV, INRA Versailles Route de Saint-Cyr RD 10, 78026 Versailles, France.
Charles-Delobel
Chrystèle
C
Geldreich
Angèle
A
Berthier
Georges
G
Boyer
Francine
F
Collonnier
Cécile
C
Coué-Philippe
Géraldine
G
Diolez
Annick
A
Duplan
Marie-Noëlle
MN
Kebdani
Naïma
N
Romaniuk
Marcel
M
Feinberg
Max
M
Bertheau
Yves
Y
D017931:DNA Primers; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D015345:Oligonucleotide Probes; D010942:Plant Viruses / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000737:chemistry; D014780:Viruses / Q000737:chemistry; D003313:Zea mays / Q000235:genetics*
D017931:DNA Primers; D018744:DNA, Plant; D004279:DNA, Viral; D015345:Oligonucleotide Probes