日本血吸虫特异的循环DNA鉴定及在诊断中初步应用

发布时间：2021-09-30 13:42

　　具有致病性的寄生蠕虫感染在全球范围内引起严重的健康问题并造成巨大的经济损失。因此,能够及时、准确地诊断蠕虫感染是控制病原传播的关键策略。以核酸检测为基础的诊断方法,主要包括聚合酶链式反应、定量qPCR、环介导等温扩增技术和重组酶聚合酶链式扩增等,均是能够有效检测蠕虫感染的方法。这些检测方法具有准确性、及时性和灵敏度高等优点。由血吸虫感染引起的血吸虫病是一个被忽视的热带性疾病,在全球范围内引起重大的公共卫生问题。血吸虫病的早期诊断和检测对该病的防控至关重要。传统的诊断方法主要包括检测粪便中的虫卵,或者通过免疫学方法检测血吸虫循环抗原。但是这些方法对血吸虫病感染早期的诊断敏感性低。循环性DNA（cfDNA）是游离于宿主体液循环中的细胞外DNA片段,它被视为一种快速、有效的检测和诊断病原体的方法。本研究中,我们探索了不同的实验动物（兔和小鼠）在感染血吸虫后线粒体循环DNA的诊断效果。首先,我们评估了三种不同的循环性DNA提取试剂盒对循环性DNA的分离效果,结果表明,QIAamp循环核酸试剂盒在提取循环性DNA的效果最好。随后,我们对日本血吸虫线粒体DNA的一些分子进行了分析,并鉴定出了线粒体... 

【文章来源】：中国农业科学院北京市

【文章页数】：63 页

【学位级别】：博士

【文章目录】：
摘要
Abstract
英文缩略表
CHAPTER 1
    1.1 Introduction
    1.2 Distribution of Schistosomes
    1.3 Life Cycle of schistosoma
    1.4 Pathogenesis of Schistosomiasis
    1.5 Cell-Free DNA and parasitic Infections
    1.6 Source of cf DNA and Its Application in Diagnosis
    1.7 Detection of schistosoma in the Circulatory System
    1.8 Problem and solution
CHAPTER 2 RECENT ADVANCES IN NUCLEIC ACID-BASED METHODS FOR DETECTION OF HELMINTH INFECTIONS AND THE PERSPECTIVE OF BIOSENSORS FOR FUTURE DEVELOPMENT
    2.2 Introduction
    2.3 Nucleic acid based methods for detection of helminth infections
        2.3.1 PCR
        2.3.2 Multiplex PCR
        2.3.3 LAMP
        2.3.4 Recombinase polymerase amplification(RPA)
    2.4 Biosensors for nucleic acid biomarker detection
        2.4.1 Electrochemical detection techniques
        2.4.2 Optical detection techniques
    2.5 Advantages and limitations of nucleic acid-based methods for detection of helminth infections
    2.6 Conclusions
CHAPTER 3 CIRCULATING CELL FREE MITOCHONDRIAL DNA FRAGMENTS ARE INFECTION
    3.2 Introduction
    3.3 Material and Methods
        3.3.1 Establishment of S.japonicum infection
        3.3.2 Praziquantel(PZQ)treatment of infected mice and blood samples collection
        3.3.3 Isolation of eggs from liver homogenate of infected mice and DNA extraction from eggs and adult S. japonicum
        3.3.4 cf DNA extraction from infected host serum
        3.3.5 Primer design and PCR amplification
        3.3.6 Gene cloning in p MD19 T vector and standard curve construction by qRT-PCR
        3.3.7 Statistical analyses
    3.4 Results
        3.4.1 Cf DNA extraction kits comparison
        3.4.2 Reference Gene of S.japonicum detected in infected rabbit serum
        3.4.3 Detection of Schistosoma japonicum cf DNA by PCR assay
        3.4.4 Detection of S.japonicum cf DNA in different week of post infection
        3.4.5 Detection of S.japonicum cf DNA in different number of cercariae of infection
        3.4.6 Efficacy evaluation after chemotherapy
        3.4.7 Characterization and copy number of target fragments
    3.5 Disscussion
    3.6 Conclusion
CHAPTER 4 DETECTION OF CIRCULATING CELL-FREE NUCLEAR DNA TO DIAGNOSE SCHISTOSOMA JAPONICUM INFECTION
    4.2 Introduction
    4.3 Material and Methods
        4.3.1 Ethics statement
        4.3.2 Establishment of S.japonicum infection and collection of samples
        4.3.3 Extraction of eggs from liver homogenate of infected mice and DNA extraction from eggs and adult S. japonicum infected host serum
        4.3.4 PCR amplification
        4.3.5 Gene cloning and standard curve construction by qRT-PCR for copy number detection
    4.4 Results
    4.5 Disscussion
    4.6 Conclusion
References
Acknowledgement
Curriculum Vitae



本文编号：3415983