Event-Specific Quantitative Detection of Nine Genetically Modified Maizes Using One Novel Standard Reference Molecule

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• 作者：Litao Yang ; Jinchao Guo ; Aihu Pan ; Haibo Zhang ; Kewei Zhang ; Zhengming Wang ; Dabing Zhang
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2007
• 出版时间：January 10, 2007
• 年：2007
• 卷：55
• 期：1
• 页码：15 - 24
• 全文大小：726K
• 年卷期：v.55,no.1(January 10, 2007)
• ISSN：1520-5118

文摘

With the development of genetically modified organism (GMO) detection techniques, the PolymeraseChain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR isthe most effective and important method for GMO quantification. An event-specific detection strategybased on the unique and specific integration junction sequences between the host plant genomeDNA and the integrated gene is being developed for its high specificity. This study establishes theevent-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specificTaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21,MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCRassays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal positionto improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certifiedreference materials of these GM maizes, one novel standard reference molecule containing all ninespecific integration junction sequences of these GM maizes and the maize endogenous referencegene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of thesemethods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies,and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template.Furthermore, nine groups of the mixed maize samples of these nine GM maize events werequantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias variedfrom 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressedas relative standard deviations was from 0.83 to 26.20%. All of these indicated that the establishedevent-specific real-time PCR detection systems and the reference molecule in this study are suitablefor the identification and quantification of these GM maizes.