基因改造玉米檢測方法之開發及尼泊爾基改玉米現狀之監測

Abstract

Genetically modified (GM) maize has been developed for human as well as livestock consumptions. For regularly monitoring and reliable control of GM maize in the market, it is essential to develop precise and accurate detection methods and set up an internationalize regulation threshold and standard. Recently, several qualitative and quantitative GM maize detection methods have been reported, validated and approved worldwide. Due to increasing number and low level presence of GM maize events; these methods must be modified, updated and/or improved regularly. Among them, multiplex PCR (mPCR) systems have been widely used for monitoring, tracing and regulating GM maize. Within the systems, event-specific strategy was found to be the most sensitive and accurate. Therefore, to fulfill labeling and traceability requirement in GM maize quality control, a current review of multiplex PCR systems was carried out and which is them applied for GM maize. An event- and construct- specific multiplex PCR system was established to detect 8 GM maize lines (Event176, Bt11, TC1705, NK603, T25, MON863, MON810 and GA21) in a reaction tube. This mPCR could detect 0.1 % GM in a proficiency test of the International Seed Testing Association (ISTA) and clearly showed the high specificity and sensitivity in GM maize detection. This system is cheap, user friendly, specific and sensitive and could substitute the existing mPCR methods based on screen-, gene-, construct- and event-specific strategies. Furthermore, the application of this technique was extended from laboratory to real field samples by monitoring the GM maize seeds status in Nepal. The aim of the research work carried out in Nepal was to check the issues of bio-conservation and bio-safety. In addition, it was also aimed to provide some useful information for establishing a trade and bio-safety threshold in Nepal. In total, 46 maize samples were collected in different locations of Nepal, where mPCR, real-time PCR and screen-specific PCR methods were applied for GM maize seed samples detection. The result showed that no maize samples were found to be contaminated with GM maize cultivars in Nepal. As this is the first report of GM maize analysis in Nepal, it would provide very basic foundation for future legal regulations, and also help for implementing quality control regulation and biological conservation of maize seeds in Nepal. Hence, for confirming the presence and/or absence of GM maize in Nepal, it is still necessary to analyse large number of maize samples covering wider areas and varieties of maize traded and/or used in Nepal. Lastly, considering the importance and usefulness of plasmid reference material in GMO quality control and method validation, a reference plasmid was constructed, containing event-specific DNA fragments from ten genetically modified maize lines (T25, MIR604, Event176, GA21, MON863, NK603, DAS-59122-7, MON810, TC1507 and Bt11) and one endogenous gene (zSSIIb), and used a fast real-time PCR system for GM maize lines quantitative analysis. The preliminary validation result showed that this system had high efficiency, specificity, sensitivity, trueness (accuracy) and could detect and quantify GM maize lines in a wide range of copy numbers from 20 to 1,000,000. Moreover, this method is considered as the latest achievement in developing plasmid reference materials including two more new events than the existing systems available in the world. As this method was based on fast real-time PCR kit and systems; reduces the volume of PCR reaction to 10μL, and only QH2O was used as non-template control; it reduces the cost and time. Therefore, system could fulfill the regularity threshold set in the world. However, further full method validation is necessary, including its limits of detection/quantification, repeatability and reproducibility. As this system was based on the event-specific strategies, it could solve the limitation of reference materials of 10 GM maize lines in the future. In sum, an event-specific mPCR method was successfully developed for GM maize analysis and its practical application was demonstrated for monitoring GM maize seed status in Nepal. In addition, an effective and efficient plasmid reference material was developed for 10 GM maize detection using a fast real-time PCR system. Therefore, this work should be interesting for most GM crops analysts, stakeholders, retailers and government agencies as well as it should benefit in precise and reliable GM maize analysis.