G9型猪轮状病毒TaqMan荧光定量RtPCR检测方的建立及初步应用.docx

CoNTENTSAbstract CoNTENTS Abstract in Chinese ．1 Abstract in English ． ． ． ． III 1 Introduction ． 1 1．1 Overview ofPoRV 1 1．1．1 Morphology,s仃Ilc臼lre and genome ofrotavirus “1 1．1．2 Physical and chemical characteristics and culture ofrotavirus ．1 1．1．3 Structural proteins 2 1．1．4 non-sn’uctIJral proteins 一3 1．1．5 Replication mechanism ．． ． ． 4 1．1．6 Epidemiology ofrotavirus ，．5 1．1．7 The manifestations and pathological changes ofrotavirus infection ．6 1．2 Detection ofrotavirus． ． ． ． 6 1．2．1 Pathogen detection technology ．7 1．2．2 Serological and immunological detection technology ．．7 1．2．3 Molecular biology detection technology ．． 8 1．3 Principle offluorescence quantitative PCR ．． ．．9 1．4 Fluorescence quantitative PCR reaction in Chemistry 9 1．4．1 Nonspecific DNA binding dye “9 1．4．2 TaqMan probe ．10 1．4．3 molecular beacon ．．10 1．4．4 Hybridization probe ．10 1．4．5 Composite probe 1 1 1．5 The application of fluorescence quantitative PCR ．1 1 1．5．1 Application ofquantitative PCR in detection ofpathogenic microorganisms 11 1．5．2 The application of quantitative detection of the expression of fluorescent quantitative PCR in cytokine gene ． ． ．．． ··1 1 1．5．3 Application offluorescence quantitative PCR in tumor research ．11 1．6 The purpose and significance ofthe study ． ． ． ．． ．．12 2 Materials and methods ．．13 2．1 Materials ．13 2．1．1 Viruses．cells and clinical samples 13 2．1．2 The reagents ．13 2．1．3 The equipments ． ． ． ． ．1 3 2．2 Methods．．． ． ．．． ． ． ． ．． ． ． ． ． ．14 2．2．1 Primers design ．14 2．2．2 The culture ofPoRV ．14 III 万方数据 东北农业大学理学硕士学位论文2．2．3 东北农业大学理学硕士学位论文 2．2．3 The determination of PoRV tissue culture infective dose ．1 4 2．2．4 n呛extraction ofvirus nucleic acid ．14 2．2．5 Preparation ofpositive standard plasmid 。16 2．2．6 The establishment offluorescent quantitative RT．PCR standard curve 。18 2．2．7 Specificity test ．1 9 2．2．8 Sensitivity test ．19 2．2．9 Repeatability test 1 9 2．2．10 The detection ofclinical samples 19 2．2．11 Infection experiments 20 2．2．1 2