动物布鲁氏菌感染重组酶聚合酶扩增检测方法的建立

发布时间：2021-12-31 18:29

　　布鲁氏菌病是一个世界性分布的人畜共患病,能够引起多种动物流产和胎儿死亡,对畜牧业造成很大的经济损失。但目前尚缺少一种快速、灵敏的检测方法用于布鲁氏菌病诊断。本试验以布鲁氏菌的bp26基因为靶基因,建立了实时重组酶聚合酶扩增（RPA）方法,并检测了不同家畜样品中的布鲁氏菌。本试验建立的RPA方法在40 ^oC条件下反应20 min,可检测到4个拷贝的含有目的片段的重组质粒。该RPA检测方法的灵敏性为94.9%,特异性为97.0%,分别略低于荧光定量PCR的91.5%和98.5%,但高于普通PCR的93.2%和97.0%。将临床样品分别用虎红平板凝胶试验与RPA方法检测,阳性检出率分别为55.6%和62.2%。该RPA方法与流产衣原体、刚地弓形虫和鼠伤寒沙门氏菌无交叉反应,说明特异性良好。本试验建立的RPA检测方法为下一步研制动物布鲁氏菌的临床检测试剂盒奠定基础。进一步,本试验将快速侧流式试纸与SYBR green I重组酶聚合酶扩增技术相结合,建立了检测布鲁氏菌的方法。针对布鲁氏菌bp26基因上的IS711区域设计引物和探针,优化反应条件。RPA在30-35℃的温度下... 

【文章来源】：中国农业科学院北京市

【文章页数】：87 页

【学位级别】：博士

【文章目录】：
摘要
abstract
Chapter Ⅰ Literature Review
    1.1.Definition the causative agents of brucellosis
    1.2.clinical manifestations and transmission
    1.3.Pathogenesis of Brucella infection
    1.4.Immune response to Brucella infection
    1.5.The genome of Brucella spp
    1.6.Diagnosis of Brucellosis:
        1.6.1.Direct methods for diagnosis of Brucella spp.infection
            1.6.1.1.Bacteriological methods
            1.6.1.2.Molecular methods
        1.6.2.In-direct diagnosis of brucellosis
            1.6.2.1.Serological tests
            1.6.2.2.Rose Bengal Plate test (RBPT)
            1.6.2.3.Complement fixation test
            1.6.2.4.Enzyme-linked Immunosorbent Assay
            1.6.2.5.Native hapten gel precipitation tests
            1.6.2.6.Fluorescence polarization assay
            1.6.2.7.Milk ring test (MRT)
            1.6.2.8.Buffered plate antigen test (BPAT)
        1.6.3.Cell-mediated immunity (CMI) based diagnosis
    1.7.Prevention,Control,and Eradication of animal brucellosis
    1.8.Recombinase Polymerase Amplification(RPA)
        1.8.1.Overview
        1.8.2.RPA reaction components
        1.8.3.RPA parameters
            1.8.3.1.Primers and Probes characteristics
            1.8.3.2.Temperature
            1.8.3.3.Incubation time
            1.8.3.4.Mixing and crowding agents
            1.8.3.5.Presence of RPA inhibitors
            1.8.3.6.Multiplex RPA
        1.8.4.Detection of RPA amplicon
            1.8.4.1.Lateral flow dipstick
            1.8.4.2.Agarose gel electrophoresis
            1.8.4.3.Bridge flocculation assay
            1.8.4.4.DVDs and low reflectivity DVDs
            1.8.4.5.Colorimetric detection
            1.8.4.6.Quantum dot(QD)barcodes
            1.8.4.7.Electrochemical transduction
            1.8.4.8.Real-time detection
            1.8.4.9.Modifications of Real-time detection
            1.8.4.10.Conclusion
Chapter Ⅱ Establishment of Recombinase Polymerase Amplification(RPA)for detection of Brucella spp.infection in animals
    2.1.Introduction
    2.2.Materials and methods
        2.2.1.Samples collection
        2.2.2.DNA extraction of Bacterial strains and samples
        2.2.3.Development and optimization of RPA
            2.2.3.1.Candidate primers and probes design
            2.2.3.2.Recombinase polymerase amplification(RPA)development and optimization
        2.2.4.Validation of RPA
            2.2.4.1.Analytical sensitivity and detection limit
            2.2.4.2.Analytical specificity
            2.2.4.3.Diagnostic sensitivity
            2.2.4.4.Recombinase polymerase amplification
            2.2.4.5.Polymerase chain reaction(PCR)
            2.2.4.6.Real time PCR
            2.2.4.7.Rose Bengal Plate test(RBPT)
        2.2.5.Statistical analysis
    2.3.Results
        2.3.1.Optimization of RPA:
        2.3.2.Analytical sensitivity and detection limit results
        2.3.3.Analytical specificity
        2.3.4.Comparison of diagnostic sensitivity and specificity of RPA,Real time PCR and PCR
        2.3.5.Comparison of diagnostic sensitivity of RPA with Rose Bengal Plate test(RBPT)
    2.4.Discussion
Chapter Ⅲ Development of the Lateral-flow dipstick combined with SYBR green I Recombinase Polymerase Amplification(RPA)for diagnosis of Brucella spp.infection
    3.1.Introduction
    3.2.Materials and Methods
        3.2.1.The samples
        3.2.2.Genomic DNA extraction
        3.2.3.Recombinant plasmid construction
        3.2.4.RPA development and optimization
            3.2.4.1.RPA primers and Probe design
            3.2.4.2.Optimization of RPA conditions
            3.2.4.3.Lateral flow dipstick(LFD)and SYBR Green I assays
            3.2.4.4.Analytical sensitivity and detection limit
            3.2.4.5.Analytical specificity
        3.2.5.Screening of field samples
        3.2.6.Real-time PCR
        3.2.7.Statistical analysis
    3.3.Results
        3.3.1.RPA optimum conditions and robustness
        3.3.2.Analytical sensitivity and detection limit
        3.3.3.Analytical specificity
        3.3.4.Screening of field samples by RPA,Real-time PCR,and PCR
    3.4.Discussion
Chapter Ⅳ Development of Duplex Recombinase Polymerase Amplification for detection of Brucella melitensis and Brucella abortus
    4.1.Introduction
    4.2.Materials and Methods
        4.2.1.Bacterial strains
        4.2.2.Collection of samples
        4.2.3.Selection of specific sequences,Bioinformatics analysis and Primers design
        4.2.4.Duplex Recombinase Polymerase Amplification(Duplex RPA)
        4.2.5.Analytical sensitivity and specificity
        4.2.6.Real-time PCR(Bounaadja et al.,2009)
        4.2.7.Multiplex AMOS PCR
        4.2.8.Screening of field samples with Duplex RPA and AMOS PCR
        4.2.9.Statistical analysis
    4.3.Results
        4.3.1.Detection of Bacterial strains by AMOS PCR
        4.3.2.Sequences alignments and primers pairs
        4.3.3.Analytical sensitivity and specificity
        4.3.4.Results of screening field samples
    4.4.Discussion
Conclusion
REFERENCES
Appendix
Acknowledgements
Author's Curriculum Vitae



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