预览加载中，请您耐心等待几秒...

IBRV与MB二温式多重PCR检测方法的建立.docx

预览
1/2
2/2

在线预览结束，喜欢就下载吧，查找使用更方便

下载提示 文本预览

如果您无法下载资料，请参考说明：

1、部分资料下载需要金币，请确保您的账户上有足够的金币

2、已购买过的文档，再次下载不重复扣费

3、资料包下载后请先用软件解压，在使用对应软件打开

IBRV与MB二温式多重PCR检测方法的建立 Title:EstablishmentofIBRVandMBDual-TemperatureMultiplexPCRDetectionMethod Abstract: Infectiousbovinerhinotracheitisvirus(IBRV)andMycoplasmabovis(MB)aretwoimportantpathogensthatprimarilyaffectcattle.Developinganefficientandaccuratedetectionmethodforthesepathogensiscrucialforcontrollingandpreventingthespreadofthediseasestheycause.Thisstudyaimstoestablishadual-temperaturemultiplexPCRdetectionmethodforIBRVandMBthatprovideshighsensitivityandspecificity. Introduction: IBRVandMBinfectionsposesignificanteconomiclossestothecattleindustryworldwide.Traditionaldetectionmethodsforthesepathogensaretime-consumingandlabor-intensive.Therefore,itisimperativetoestablisharapidandreliablediagnosticmethodforsimultaneousdetectionofIBRVandMB.MultiplexPCR,whichallowsmultipletargetgenestobeamplifiedsimultaneously,hasshowngreatpotentialinthefieldofpathogendetection.Thisstudyaimstoestablishadual-temperaturemultiplexPCRdetectionmethodforIBRVandMB. Methods: 1.Primerdesign:SpecificprimersforIBRVandMBtargetgenesweredesignedusingbioinformaticstools.Theprimersweredesignedtoamplifydifferentregionsofthetargetgenestoensurespecificity. 2.DNAextraction:DNAsampleswereextractedfromclinicalsamples(nasalswabs,lungtissues,orbloodsamples)usingacommercialDNAextractionkit. 3.MultiplexPCRoptimization:Theoptimalreactionconditions,includingprimerconcentrations,magnesiumionconcentration,cyclingparameters,andenzymeconcentration,weretestedandoptimizedusingpureDNAtemplates. 4.Sensitivityandspecificityevaluation:Thesensitivityandspecificityofthedual-temperaturemultiplexPCRmethodwereevaluatedusingknownpositiveandnegativesamples.TheresultswerecomparedwiththoseofconventionalPCRandsequencingmethods. Results: 1.Primerdesign:SpecificprimerstargetingtheIBRVandMBgenesweresuccessfullydesignedbasedongenesequencesavailableinpublicdatabases. 2.MultiplexPCRoptimization:Theoptimalreactionconditionsweredeterminedasfollows:denaturationat95°Cfor2minutes,followedby35cyclesofdenaturationat95°Cfor30seconds,annealinga