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猪圆环病毒3型实时荧光定量PCR方法的建立和应用.docx

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猪圆环病毒3型实时荧光定量PCR方法的建立和应用 Title:EstablishmentandApplicationofReal-timeFluorescentQuantitativePCRMethodforPorcineCircovirusType3 Abstract: PorcineCircovirusType3(PCV3)isanewlydiscoveredporcinepathogen,whichhascausedconsiderableeconomiclossesintheswineindustryworldwide.TherapidandaccuratediagnosisofPCV3iscrucialforeffectivepreventionandcontrol.Thispaperaimstoestablishareal-timefluorescentquantitativePCRmethodforPCV3andexploreitsapplicationinthediagnosisofPCV3infection. Introduction: PCV3isasmall,non-enveloped,single-strandedDNAvirusthatbelongstotheCircoviridaefamily.Itisassociatedwithavarietyofclinicalconditionsinpigs,includingreproductivedisorders,respiratorydiseases,anddevelopmentofmultisystemicwastingsyndrome.Todate,thereislimitedliteratureonthedevelopmentofPCR-basedmethodsspecificallytargetingPCV3.Therefore,theestablishmentofareal-timefluorescentquantitativePCRmethodforPCV3willgreatlycontributetoitsdiagnosisandsurveillance. Methodology: 1.DesignofPCRprimersandprobes:PrimersandprobesspecifictothePCV3genomeweredesignedbasedonpublishedPCV3sequences.Bioinformaticstoolswereusedtoensuretheirspecificityandavoidpotentialcross-reactivitywithotherpathogens. 2.DNAextraction:ViralDNAwasextractedfromPCV3-positivesamplesusingacommercialDNAextractionkitfollowingthemanufacturer'sinstructions. 3.Real-timePCRreaction:Thereal-timePCRreactionmixturewasprepared,containingtheextractedDNA,specificprimers,andprobes,aswellasaPCRmastermixthatincludedpolymerase,dNTPs,andothernecessarycomponents.Thereactionwasperformedusingareal-timePCRmachine,andfluorescencesignalsweremonitoredduringtheamplificationprocess. 4.Standardcurveconstruction:Aseriesof10-folddilutionsofknownPCV3viralDNAconcentrationwereusedtoconstructastandardcurve.Thiscurvewasusedtodeterminetheviralloadinthetestedsamples. 5.Sensitivityandspecificityevaluation:ThesensitivityandspecificityofthedevelopedPCRmethodwereevaluatedusingapanelofPCV3-positiveandPCV3-negativesamples,respectively.Theresultswerecomparedwiththoseobtainedusingothe