甘蔗桿狀病毒(Sugarcane bacilliform viruses,SCBVs)屬于花椰菜花葉病毒科Caulimoviridae桿狀DNA病毒屬Badnavirus,是危害甘蔗的主要病毒之一,在世界各主要甘蔗產(chǎn)區(qū)均有發(fā)現(xiàn),嚴(yán)重影響甘蔗的產(chǎn)量和品質(zhì)。因此,急需建立一種快速、靈敏、特異的檢測(cè)方法,為阻止SCBV病毒跨區(qū)域傳播,為該病害隔離檢疫和健康脫毒種苗質(zhì)量監(jiān)控提供關(guān)鍵技術(shù)支撐。本研究針對(duì)2012年國(guó)際病毒分類委員會(huì)ICTV公布的兩個(gè)SCBV病毒種Sugarcane bacilliform IM virus(SCBIMV,NC₀03031)和Sugarcane bacilliform MO virus(SCBMOV,NC₀08017)的反轉(zhuǎn)錄酶/RNA 酶 H(RT/RNase H)序列分別設(shè)計(jì)了一套特異性引物和探針,通過(guò)優(yōu)化反應(yīng)體系和條件,建立了靈敏度高、特異性強(qiáng)、重現(xiàn)性好的TaqMan實(shí)時(shí)熒光定量PCR(qPCR)檢測(cè)方法。該方法以重組質(zhì)粒PMD18T-IM(SCBIMV)和pSCBV20(SCBMOV)重組質(zhì)粒DNA為模板(109-...

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【學(xué)位級(jí)別】：碩士

【文章目錄】：
摘要 Abstract 1 Introduction
1.1 Sugarcane production in China
1.2 Sugarcane viral diseases in China
    1.2.1 Overview of viral diseases
    
1.2.1.1 Viral diseases controls
    
1.2.1.2 Usages of disease-resistance cultivars
    
1.2.1.3 Tissue culture
    
1.2.1.4 Transgenic plants
1.3 Sugarcane bacilliform virus
    1.3.1 Geographical distribution
    1.3.2 Symptoms
    1.3.3 Host range
    1.3.4 Transmission
    1.3.5 Genome characteristics
    1.3.6 Variability and Molecular identification
    1.3.7 Detection methods of SCBV and their limitations
    
1.3.7.1 Symptomatology
    
1.3.7.2 Electron microscopy
    
1.3.7.3 Serology
    
1.3.7.4 Polymerase chain reaction (PCR)
    
1.3.7.5 Immunocapture PCR (IC-PCR)
1.4 SCBV disease control
1.5 Purpose and objective of study 2 Materials and Methods
2.1 Leaves sampling
2.2 Reagents and kits
2.3 DNA Isolation and purification
2.4 Primer and probe design
2.5 Plasmids generation
2.6 Plasmid extraction and dilution
2.7 qPCR assay
2.8 qPCR standard curve construction
2.9 Sensitivity test of the qPCR
2.10 Specificity test of the qPCR
2.11 Conventional PCR
2.12 PCR products purification
2.13 PCR products cloning and sequencing
2.14 Field samples detection for SCBV
2.15 Sequence alignment
2.16 Phylogenetic analysis
2.17 Sequence identity and genetic distance analysis 3 Results
3.1 Primer specificity analysis in-silico
3.2 Efficiency of Real-Time qPCR Assay
3.3 Sensitivity of the qPCR
3.4 Specificity of the qPCR
3.5 Field samples detection for SCBV
3.6 SCBV genotypes identification
    3.6.1 Phylogenetic grouping
    3.6.2 Sequences identity
    3.6.3 Sequence genetic distance 4 Discussion and Conclusion
4.1 qPCR assays is more sensitive and efficient for SCBV detection
4.2 Highly genetic variation among the SCBV genotypes/strains
4.3 Quarantine inspection is necessary in plant cutting exchange
4.4 Conclusions References Published Papers Appendices Acknowledgement



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