Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.
In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.
Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety, Fuzhou University, Fuzhou, P. R. China.
Longhua
L
Guo
Bin
B
Qiu
Yuwu
Y
Chi
Guonan
G
Chen
D000818:Animals; D018744:DNA, Plant / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D030781:Organisms, Genetically Modified / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018744:DNA, Plant