小鹅瘟病毒荧光PCR检测方法的建立

2010.05GooseParvovirusGPV、[1]。4～20[2-4]。[5-7]。PCR[8]。TaqmanGPVNSPCRGPV。、、、、GPV。11.1.1GPV。1.1.2pMD18-TSimpleVector、DH5αDNA、、。1.2.1GenBankGPVNSOligo6.0Primer5.0。P15′-TCTGCTCTCAGAGAGAACG-3′P25′-ATTACCCACTCCATCGGC-3′P35′-AGTGATTTGGCTGCCCCTTTA-3′P45′-TCTTTGCCGCTCTGTTGGA-3′fam-ATCCTCAAGCATCAACTGTGGCACC。1.2.2DNAGPV59～11SPF0.2ml7d48h。48～168h4℃。。0.2ml/。DNADNA。1.2.3PCRDNAPCR。P1、P250μlPCR10×buffer5μldNTP4μlP1、P21μlDNA1μlExTaq0.5μlddH2O37.5μl。95℃5min94℃1min56℃1min72℃2min30PCR1211211.1101612.110016S852.65+9.2B1672-9692(2010)05-0051-04[]GenBankGPVNSPCRNS、TaqmanPCR。GPV。[]TaqmanPCR!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!512010.0572℃10min。10μlPCR1.0%。1.2.4PCRPCR10μlPCR2590bpPMD18-TSimpleVectorDH5аAMPLB37℃AMPLB37℃。PCR。。NCBI100%。1.2.5PCRP3、P425μlPCR10×buffer1μldNTP1μlP3、P41μlDNA0.5μlrTaq0.25μl1μlddH2O18.25μl。95℃3min95℃10s57℃50s72℃15s4072℃10min。。、。1.2.6DNA1.2.4100%DNANDA。1.2.7DNA1091.0×100～1.0×10-8PCR10/PCR。2PCR2594bp1。PCRPCRpMD18-T-GOVNSPCR1%22。NDA8CT3。8NDACt0.99845。PCRGPV、GPMV、、、。1PCR2PCR3PCR522010.056。3PCRPCR、、、。Taqman[9]。GPVNSGPVTaqmanGPVPCR。1.0×10-8。GPV、GPMV、、。。[]5PCR1.0×100～1.0×10-8。64PCR1.0×100～1.0×10-8。532010.052009。100%77.4%。1。、。2d。。。21~2d。。39℃37℃。474。2、、。325%10ml5%10ml2/d7d。。、、。。4、、。。。EstablishmentandapplicationofFL-PCRtodetectgooseparvovirusviruseLongZhen1LinYing2ZhangTongyuan1ShengMingwei1GengQinghua1ZhaoYujun11.ShenyangAgricultureUniversityLiaoningShenyang110161;2.ShenyangEntry-ExitInspectionQuarantineLiaoningShenyang110016!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!54