A Reverse Transcriptase-PCR Assay for Detecting Filarial Infective Larvae in Mosquitoes 英文参考文献.docVIP

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A Reverse Transcriptase-PCR Assay for Detecting Filarial Infective Larvae in Mosquitoes 英文参考文献.doc

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A Reverse Transcriptase-PCR Assay for Detecting Filarial Infective Larvae in Mosquitoes 英文参考文献

AReverseTranscriptase-PCRAssayforDetectingFilarial InfectiveLarvaeinMosquitoes SandraJ.Laney1*,CaitlinJ.Buttaro1,SabatoVisconti1,2,NilsPilotte1,RedaM.R.Ramzy3,GaryJ.Weil4, StevenA.Williams1,5 1DepartmentofBiologicalSciences,SmithCollege,Northampton,Massachusetts,UnitedStatesofAmerica,2AmherstCollege,Amherst,Massachusetts,UnitedStatesof America,3NationalNutritionInstitute,Cairo,Egypt,4InfectiousDiseaseDivision,DepartmentofInternalMedicine,WashingtonUniversitySchoolofMedicine,St.Louis, Missouri,UnitedStatesofAmerica,5PrograminMolecularandCellularBiology,UniversityofMassachusetts,Amherst,Massachusetts,UnitedStatesofAmerica Abstract Background: Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoesthatcontainanystageoftheparasiteandinfectivemosquitoesthatharborthirdstagelarvae(L3)capableof establishingnewinfectionsinhumans.WenowreportdevelopmentofamolecularL3-detectionassayforBrugiamalayiin vectorsbasedonRT-PCRdetectionofanL3-activatedgenetranscript. Methodology/PrincipalFindings:CandidategenesidentifiedbybioinformaticsanalysisofESTdatasetsacrosstheB.malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolatedfrominfectedmosquitoes.Mosquitoeswerecollecteddailyfor14daysafterfeedingonmicrofilaremiccatblood. RT-PCRwasperformedwithprimersetsthatwerespecificforindividualcandidategenes.Manypromisingcandidateswith strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100,whichencodesaparticularformofcollagen)wasonlydetectedinmosquitoesthatcontainedL3larvae.Thisassay detectsasingleL3inapoolof25mosquitoes. Conclusions/Significance:ThisL3-activatedgenetranscript,combinedwithacontroltranscript(tph-1,accession#U80971) thatisconstitutivelyexpressedbyallvector-stagefilariallarvae,canbeusedtodetectfilarialinfectivityinpoolsofmosquito vectors.Thisgeneralapproach(detectionofstage-speci