Event-specific qualitative and quantitative polymerase chain reaction analysis for genetically modified canola T45.
Polymerase chain reaction (PCR) methods have been the main technical support for the detection of genetically modified organisms (GMOs). To date, GMO-specific PCR detection strategies have been developed basically at four different levels, such as screening-, gene-, construct-, and event-specific detection methods. Event-specific PCR detection method is the primary trend in GMO detection because of its high specificity based on the flanking sequence of exogenous integrant. GM canola, event T45, with tolerance to glufosinate ammonium is one of the commercial genetically modified (GM) canola events approved in China. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of T45 canola was cloned and revealed by means of TAIL-PCR. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence, and qualitative and quantitative TaqMan real-time PCR detection assays employing these primers and probe were developed. In qualitative PCR, the limit of detection (LOD) was 0.1% for T45 canola in 100 ng of genomic DNA. The quantitative PCR assay showed limits of detection and quantification (LOD and LOQ) of 5 and 50 haploid genome copies, respectively. In addition, three mixed canola samples with known GM contents were detected employing the developed real-time PCR assay, and expected results were obtained. These results indicated that the developed event-specific PCR methods can be used for identification and quantification of T45 canola and its derivates.
Yang
Litao
L
SJTU-SIBS-PSU Joint Center for Life Sciences, Key Laboratory of Microbial Metabolism, Ministry of Education, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People's Republic of China.
Pan
Aihu
A
Zhang
Haibo
H
Guo
Jinchao
J
Yin
Changsong
C
Zhang
Dabing
D
D001483:Base Sequence; D029688:Brassica napus / Q000235:genetics*; D018744:DNA, Plant / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control
D018744:DNA, Plant