g9型猪轮状病毒taqman荧光定量rtpcr检测方法的建立及初步应

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1、CoNTENTSAbstractinChinese．1AbstractinEnglish．．．．III1Introduction．11．1OverviewofPoRV11．1．1Morphology,s仃Ilc臼lreandgenomeofrotavirus“11．1．2Physicalandchemicalcharacteristicsandcultureofrotavirus．11．1．3Structuralproteins21．1．4non-sn’uctIJralproteins一31．1．5Replicationmechanism．．．．41．1．6Epidemiologyof

2、rotavirus，．51．1．7Themanifestationsandpathologicalchangesofrotavirusinfection．61．2Detectionofrotavirus．．．．61．2．1Pathogendetectiontechnology．71．2．2Serologicalandimmunologicaldetectiontechnology．．71．2．3Molecularbiologydetectiontechnology．．81．3PrincipleoffluorescencequantitativePCR．．．．91．4Fluorescen

3、cequantitativePCRreactioninChemistry91．4．1NonspecificDNAbindingdye“91．4．2TaqManprobe．101．4．3molecularbeacon．．101．4．4Hybridizationprobe．101．4．5Compositeprobe111．5TheapplicationoffluorescencequantitativePCR．111．5．1ApplicationofquantitativePCRindetectionofpathogenicmicroorganisms111．5．2Theapplicati

4、onofquantitativedetectionoftheexpressionoffluorescentquantitativePCRincytokinegene．．．．．··111．5．3ApplicationoffluorescencequantitativePCRintumorresearch．111．6Thepurposeandsignificanceofthestudy．．．．．．．122Materialsandmethods．．132．1Materials．132．1．1Viruses．cellsandclinicalsamples132．1．2Thereagents．1

5、32．1．3Theequipments．．．．．132．2Methods．．．．．．．．．．．．．．．．．142．2．1Primersdesign．142．2．2ThecultureofPoRV．14III万方数据东北农业大学理学硕士学位论文2．2．3ThedeterminationofPoRVtissuecultureinfectivedose．142．2．4n呛extractionofvirusnucleicacid．142．2．5Preparationofpositivestandardplasmid。162．2．6Theestablishmentoffluorescentqua

6、ntitativeRT．PCRstandardcurve。182．2．7Specificitytest．192．2．8Sensitivitytest．192．2．9Repeatabilitytest192．2．10Thedetectionofclinicalsamples192．2．11Infectionexperiments202．2．12DetectionofdetoxificationofpigletsbythefluorescentquantitativePCR。202．2．13Preparationoftissuesections212．2．14Dyeing．．213Resu

7、ltsandanalysis．223．1TheresultsofPoRVtissuecultureinfectivedose．．223．2Theextractionofvirusnucleicacid．．223．3TheestablishmentoffluorescentquantitativeImPCRstandardcurve。223．4Theresultsofspecificitytest233．5Theresultsofsensitiv