布魯氏菌病是一個(gè)世界性分布的人畜共患病,能夠引起多種動(dòng)物流產(chǎn)和胎兒死亡,對畜牧業(yè)造成很大的經(jīng)濟(jì)損失。但目前尚缺少一種快速、靈敏的檢測方法用于布魯氏菌病診斷。本試驗(yàn)以布魯氏菌的bp26基因?yàn)榘谢?建立了實(shí)時(shí)重組酶聚合酶擴(kuò)增（RPA）方法,并檢測了不同家畜樣品中的布魯氏菌。本試驗(yàn)建立的RPA方法在40 ^oC條件下反應(yīng)20 min,可檢測到4個(gè)拷貝的含有目的片段的重組質(zhì)粒。該RPA檢測方法的靈敏性為94.9%,特異性為97.0%,分別略低于熒光定量PCR的91.5%和98.5%,但高于普通PCR的93.2%和97.0%。將臨床樣品分別用虎紅平板凝膠試驗(yàn)與RPA方法檢測,陽性檢出率分別為55.6%和62.2%。該RPA方法與流產(chǎn)衣原體、剛地弓形蟲和鼠傷寒沙門氏菌無交叉反應(yīng),說明特異性良好。本試驗(yàn)建立的RPA檢測方法為下一步研制動(dòng)物布魯氏菌的臨床檢測試劑盒奠定基礎(chǔ)。進(jìn)一步,本試驗(yàn)將快速側(cè)流式試紙與SYBR green I重組酶聚合酶擴(kuò)增技術(shù)相結(jié)合,建立了檢測布魯氏菌的方法。針對布魯氏菌bp26基因上的IS711區(qū)域設(shè)計(jì)引物和探針,優(yōu)化反應(yīng)條件。RPA在30-35℃的溫度下... 

【文章來源】：中國農(nóng)業(yè)科學(xué)院北京市

【文章頁數(shù)】：87 頁

【學(xué)位級別】：博士

【文章目錄】：
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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