Event-specific qualitative and quantitative PCR methods for the detection of genetically modified rapeseed Oxy-235.
Oxy-235 is an oxynil-tolerant genetically modified rapeseed approved for commercialized planting in Canada. The aim of this study was to establish event-specific qualitative and quantitative detection methods for Oxy-235. Both the 5'- and 3'-junction sequences spanning the plant DNA and the integrated gene construct of the Oxy-235 event were isolated, sequenced and analyzed. A 1298-bp deletion of the rapeseed genomic DNA that showed a high similarity to the mRNA sequence of Arabidopsis thaliana was found in the integration site of the insert DNA. Event-specific qualitative PCR methods were established, with one method producing a 105-bp product specific for the 5'-integration junction and the other method producing a 124-bp product specific for the 3'-junction. The absolute detection limits for the qualitative PCR were determined to be 100 initial template copies for the 5'-junction and ten for the 3'-junction. Quantitative methods were also developed that targeted both of the junction fragments. The limit of detection of the quantitative PCR analysis was ten initial template copies for either the 5'- or 3'-junction, while the limit of quantification was determined to be approximately 50 initial template copies. The real-time PCR systems so established were examined with two mixed rapeseed samples with known Oxy-235 contents and found to obtain the expected results.
Wu
Gang
G
Oilcrops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan, 430062, China.
Wu
Yuhua
Y
Xiao
Ling
L
Lu
Changming
C
D001483:Base Sequence; D029741:Brassica rapa / Q000235:genetics*; D017931:DNA Primers; D015342:DNA Probes; D018744:DNA, Plant; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D015342:DNA Probes; D018744:DNA, Plant