临床常见细菌和真菌快速检测方法的初步研究

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1、硕士学位论文PreliminarystudiesontherapiddetectionofclinicalcommubacteriandfunffCUnicalC0onacteriaanlunglName：HartHuiSupervisor：ProfessorWuBingyiABSTRACTBacterialandfungalinfectionsareclinicalcommoninfectiousdiseases埘tllhi曲morbidityandmortality,whichthreatenl

2、ivesofpatientswithoutearlydiagnosisandtreatment．Thetraditionalmethodsincludingmicrobialculture，biochemicaltestsandimmunoassaywerelackofsensitivityandspecificityanddidnotdiagnosepathogenicinfectionrapidlyandaccurately．Soitisveryimportanttoestablishaquic

3、kdiagnosismethod．Inthisstudy,molecularbiologymethodswereestablishedforrapiddiagnosisofcommonbacteriaandfungiinclinicalinfectionsamples．Theaimsaretoprovidepotentialguidestorationalantibioticadministrationinclinicalpractice．Part1Establishmentofreal·timeP

4、CRfordetectionofbacteriaandfungisimultaneouslyObjeetive：TaqManprobe—basedreal-timePCRwasestablishedfordetectionanddifferentiationofgram—positivebacteria，gram—negativebacteriaandfungiinfectionssimultaneousinonetest．Itwouldmakeitpossibletodiagnosepathoge

5、nsatanearlystage．Methods：Twentybacterial16SrRNAgeneandsevenfungal18SrRNAsequenceswereobtainedfromGenBankandaligned、肮tIlsequencestoidentifyrespectiveconservedABSTRACTsequencesus吨theprogramClustalX．Accordingtogeneralprincipleofdesign，bacterialuniversalpr

6、imersandgram—staintypingprobesweredesignedbasedonbacterialconservedsequences，andfungaluniversalprimersanduniversalprobeweredesignedbasedonconservedregionsbythePrimerExpress2．0andOligo6．0software．Thegram·positiveprobeand劬galprobewerelabeledwith6-carboxy

7、fluoreseein(FAM)reporterdyeatthe5'-terminal，gram—negativeprobewaslabeledwithHEX．TheprobeswerelabeledwithfluorescentquencherTAMRAatthe3'-terminal．A229bpfragmentWasamplifiedbybacterialprimersand276bpamplifiedbyfungalprimers．Afteroptimizationofreactionsys

8、tem，thebacterialgram-positiveandgram—negativeprobeswereplacedinareactiontube，andfungaluniversalprobeinanothertube，thusPCRamplificationofbacterialandfungaltargetDNAaretwoparallelreactions．Eachassayincludednegative，positiveandblankcontrol