侵袭性曲霉菌感染实时荧光定量pcr检测新技术的建立与临床应用

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1、中文摘要课题的创新之处(1)通过特异性引物、探针的设计，建立具有自主知识产权的曲霉菌荧光定量PCR检测新技术。(2)建立适合临床实验室采用的提取曲霉菌DNA方法。(3)曲霉菌荧光定量PCR技术可同时检测包括烟曲霉菌在内的5种常见曲霉菌(黄曲霉、士曲霉，黑曲霉、构巢曲霉)，覆盖面全。(4)比较荧光定量PCR方法与血清学方法对临床病例标本诊断效能的差异，对荧光定量PCR方法进行了较全面的诊断效能评价。关键词：曲霉菌侵袭性曲霉菌病荧光定量PCR诊断IV硕士学位论文EstablishmentandclinicalapplicationofReal-timePCRforinv

2、asiveaspergillosisdetectioninbloodName：ZhangMengyuSupervisor：WangQianABSllRACTBackground：Invasiveaspergillnsis(IA)hasbecomeamajorcan$eofdc抽duetoinfectioninimmunocompromisedpatients．Itremainsahighmortality(40％一100％)amongthosetreatedforhaematologiealmalignancyandthosechemotherapyandrecei

3、vingasolidorgantransplant．Thehighmortalityisdueinparttodifficultiesindiagnosisbecausethetraditionaltechniquesinlaboratorylacksensitivityandwastetime．InrecentyearsanewdiagnositicassayhasfocusonthedetectionofcirculatingmarkersincludinggcuomicfungalDNAandfllngalantigen．Objective：Todevelop

4、areal·timePCRassayforrapiddetectingaspergillusinbloodusingself-designedprimersandprobe．ToestablishastandardizedmethodfortheextractionoffungalDNAfromclinicalspecimens．ThereisaneedtodevelopastandardprotocolforthedetectionofAspergillusspp．ThetestwillbeevaluatedbyDNAisolationfrompatientswh

5、owereathighriskofinvasiveasper翻llosisinfoaion．Theresultofclinicalstudysupposedtobethegnidlinefordrawingblood．Methods：(i．)Basedonthe18sRNA,5．8sRNA,28sRNA,ITSI、ITSIIgenesequenceofaspergillns，severalrelativefongiesandhumanbeinginthegenebank．Onepairof垒璺§!墨垒!!。primersandoneprobeweredesigned

6、usingABIPrimerExpress2．0·Wedesignedanotherpairofprimernearthetargetamplificationandobtainanucleotidesequence，thelengthofwhichwas200-300bp．then，thenucleotidesequencewasclonedintothevectorpMDl9．T．Therecombinantvectorwasusedtoestablishthestandardcurvewhichcanquantifytheinitialfungalburden

7、ofthesample．ThestudyalsoincludedtheapproachtofindthebestwayofextractingthefungalDNAfromclinicalsample，thesensitivity,specificityandreproducibility．@wecollected232samplesof71patientsathighriskofIAwhowereonceinNanFanghospitalfromApril．2006toFebruary．2007．Thesensitivity,specificity,accu