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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 胡凱康(Kae-Kang Hwu),張孟基(Men-Chi Chang) | |
dc.contributor.author | Hari Kumar Shrestha | en |
dc.contributor.author | 哈利 庫瑪若 薛瑞斯塔 | zh_TW |
dc.date.accessioned | 2021-06-15T05:13:15Z | - |
dc.date.available | 2012-08-05 | |
dc.date.copyright | 2010-08-05 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-22 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46517 | - |
dc.description.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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:13:15Z (GMT). No. of bitstreams: 1 ntu-99-D93621103-1.pdf: 11838770 bytes, checksum: 0ca5d805998f5f111e7f3c7b21b94c32 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | Declaration ii
Dedication iii Abstract iv Acknowledgements vii Table of Contents x Tables xiv Figures xv Appendixes xvii Abbreviations xviii Chapter 1 1 General Introduction 1 1.1. Agriculture and Maize 1 1.2. Biological Background of Maize 2 1.3. Global Status of Maize (Area, Production and Yield) 3 1.4. Genetically Modified Organisms (GMOs) 4 1.5. Global Status of GM Crops including GM Maize 6 1.6. Impact of GM Maize 7 1.6.1. Potential benefits of GM crops 8 1.6.2. Risk and challenges to the environmental and maize growing farmers 10 1.6.3. Risk assessment 15 1.7. Legislation of GMOs 16 1.7.1. European Union (EU) (process-based) 17 1.7.2. USA (product-based/vertical) 18 1.7.3. Canada 19 1.7.4. Australia and NewZealand 19 1.7.5. Japan 19 1.7.6. Taiwan 19 1.8. Detection of GMOs 20 1.8.1. Protein-based method 20 1.8.2. DNA based methods 21 1.8.3. Strategies of PCR system 21 1.8.4. Muliplex PCR system 24 1.9. Method Validation 25 1.10. Aims of this Study 27 1.11. Experimental Design and Format (Organization) of Dissertation 28 Chapter 2 29 Advances in Detection of Genetically Modified Crops by Multiplex PCR Methods 29 Abstract 29 2.1. Introduction 30 2.2. Advantages and Limtations of mPCR Methods 33 2.3. Factors Affecting mPCR 34 2.4. Qualitative and Quantitative Analysis of GM Crops using mPCR or Multiplex Real-Time PCR (mrt-PCR) Method 34 2.5. Other mPCRs coupled with Different Methods 35 2.5.1. mPCR coupled with capillary gel electrophoresis (mPCR-CGE) 36 2.5.2. mPCR coupled with microarray 36 2.5.3. Multiplex ligase chain reaction coupled with mPCR method (mPCR-mLCR) 37 2.5.4. DNA biosensor coupled with biotinlyted mPCR 39 2.5.5. Continuous-flow PCR reaction microfluidics 40 2.6. Future Challenges in GM Crops Detection System 41 2.6.1. Monitoring or tracing GM crops with stacked traits 41 2.6.2. Detecting unknown (new) or unexpected DNA sequence in GM crops 43 2.6.3. Standarization of detection procedure, including availability of reference materials 43 2.6.4. Time and cost consuming of lengthy validation process 44 2.6.5. Inhibition effect or unequal amplification of PCR products in GM crops detection 44 2.6.6. Lack of up-date database and information of GM crops or derived products 45 2.7. Conclusions 46 Chapter 3 47 Simultaneous Detection of Eight Genetically Modified Maize Lines Using a Combination of Event- and Construct-Specific Multiplex-PCR Technique 47 Abstract 47 3.1. Introduction 48 3.2. Materials and Methods 52 3.2.1. Materials 52 3.2.2. Genomic DNA isolation, purity and concentration determination 52 3.2.3. Preparation of test samples 53 3.2.4. DNA sequence analysis and oligonucleotide primer designing 53 3.2.5. Optimization and validation of PCR conditions 54 3.3. Results and Discussions 56 3.3.1. Internal sample control and event-specific primers designing 56 3.3.2. Primer specificity validation for singlet-PCR 56 3.3.3. Specificity test of multiplex-PCR 58 3.3.4. Limit of detection (LOD) 58 3.3.5. ISTA proficiency test 59 3.4. Conclusions 61 3.5. Acknowledgements 61 Chapter 4 62 Absence of Genetically Modified Maize (Zea Mays L.) in Seed Samples from Nepal 62 Abstract 62 4.1. Introduction 63 4.2. Materials and Methods 66 4.2.1. Seed and grain materials 66 4.2.2. Standard DNA samples 66 4.2.3. Isolation and quantification of genomic DNA 67 4.2.4. Primers and probes 67 4.2.5. Multiplex PCR 68 4.2.6. Real-time PCR 69 4.2.7. Screen-specific PCR 69 4.3. Results and Discussions 70 4.3.1. Specificity and sensitivity of mPCR in detecting GM maize 70 4.3.2. Detection of GM maize by mPCR 71 4.3.3. Real-time PCR analysis of samples with suspected GM maize 71 4.3.4. GM maize screening by screen-specific single PCR 72 4.4. Conclusions 74 4.5. Acknowledgements 76 Chapter 5 77 Development of an Event-Specific Plasmid Reference Material for the Quantitative Analysis of Ten Genetically Modified Maize Lines Based on Fast Real-Time PCR System 77 Abstract 77 5.1. Introduction 78 5.2. Materials and Methods 83 5.2.1. Seed materials 83 5.2.2. DNA extraction and purification 83 5.2.3. Oligonucleotide primers and probe 84 5.2.4. Qualitative PCR for primers specificity analysis 86 5.2.5. Construction of the standard reference molecule 86 5.2.6. Cloning of 11 targets into pGEM-T Easy Vector and transformation into E.coli 89 5.2.7. Real-time PCR for quantitative analysis 90 5.3. Results and Discussions 92 5.3.1. Designing of the event-specific primer and probes 92 5.3.2. Specificity analysis of event-specific primers and probes 93 5.3.3. Construction of reference materials 94 5.3.4. Sequence analysis and fast real-time PCR 96 5.3.5. Method validation 97 5.4. Conclusions 102 Chapter 6 103 Overall Conclusions and Future Perspectives 103 References 107 Tables 119 Figures 139 Appendixes 166 Curriculum Vitae 193 | |
dc.language.iso | en | |
dc.title | 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 | zh_TW |
dc.title | Development of Genetically Modified (GM) Maize (Zea mays L.) Detection Methods and Monitoring GM Maize Status in Nepal | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 劉麗飛(Li-Fei Liu),潘子明(Tzu-Ming Pan),黃鵬林(Pung-Ling Huang),吳明哲(Min-Tze Wu),高文彥(Wen-Yen Kao) | |
dc.subject.keyword | 基因改良玉米, | zh_TW |
dc.subject.keyword | Genetically modified (GM) maize (Zea mays L.),PCR strategies,multiplex-PCR (mPCR),real-time PCR (rtPCR),transgene integration sequence,stacked traits,plasmid reference material (PRM),genomic reference material (GRM),certified reference material (CRM), | en |
dc.relation.page | 205 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-07-23 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農藝學研究所 | zh_TW |
顯示於系所單位: | 農藝學系 |
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