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1、浙江農(nóng)業(yè)學(xué)報ActaAgriculturaeZhejiangensis20(5):328~332,2008豬繁殖與呼吸綜合征病毒熒光定量PCR檢測方法的建立1,*1,21112徐輝,方立,陳偉杰,趙靈燕,倪柏鋒,方維煥(1浙江省畜牧獸醫(yī)局,浙江杭州310020;2浙江大學(xué)動物預(yù)防醫(yī)學(xué)研究所,浙江省重點實驗室,浙江杭州310029)摘要:針對豬繁殖與呼吸綜合征病毒(Porcinereproductiveandrespiratorysyndromevirus,PRRSV)N基因保守序列設(shè)計引物與TaqMan探針,在建立常規(guī)PCR的基礎(chǔ)上,設(shè)計并優(yōu)化了TaqMa
2、n熒光定量PCR方法用于PRRSV核酸的檢測。應(yīng)用新建立的熒光定量PCR方法檢測其它主要豬病病原,無任何非特異性反應(yīng);針對PRRSV1陽性克隆質(zhì)粒的檢測靈敏度可以達到1.210copy/ml,比常規(guī)PCR高100倍;通過對48份臨床疑似樣本的檢測表明有29份樣本為PRRSV陽性,與巢式PCR檢測結(jié)果一致,而常規(guī)PCR只能檢出其中19份陽性樣本,靈敏度優(yōu)于常規(guī)PCR方法。結(jié)果表明,試驗建立的PRRSVTaqMan熒光定量PCR方法具有良好的特異性、敏感性和重復(fù)性,可為PRRSV臨床診斷提供一個更為有效的方法。關(guān)鍵詞:豬繁殖與呼吸綜合征病毒;熒光定量PCR;Taq
3、Man探針中圖分類號:S828文獻標識碼:A文章編號:1004-1524(2008)05-0328-05QuantitativeTaqManrea-ltimePCRfordetectionofporcinereproductiveandresp-iratorysyndromevirus1,*1,21112XUHui,FANGLi,CHENWe-ijie,ZHAOLing-yan,NIBa-ifeng,FANGWe-ihuan12(ZhejiangBureauofAnimalHusbandryandVeterinaryMedicine,Hangzho
4、u310020,China;ZhejiangProvincialKeyLab-oratoryofPreventiveVeterinaryMedicine,InstituteofPreventiveVeterinaryMedicine,ZhejiangUniversity,Hangzhou310029,China)Abstract:ATaqMan-basedrea-ltimePCRassaywasdevelopedtorapidlydetecttheporcinereproductiveandrespiratorysyndromevirus(PRRSV).Prim
5、ersandprobespecifictotheconservedregionofthePRRSVNgenewereselected,andthereactivesystemandconditionswereoptimizedtoimprovethesensitivity,specificityandrepetitionoftheassay.Theresultsshowedthattherea-ltimePCRassaywasspecificandtherewerenocrossreactionswithothercommonporcineviruses.The
6、1sensitivityoftheassaywas1.210copy/mlofplasmidcodingpartialPRRSVNgene,whichis100timeshigherthancon-ventionalPCR.IttooknomorethanthreehoursfromviralRNAextractiontocompletetherea-ltimePCR,andtheassaywassimpleandhadgoodrepeats.Amongtissuesamplesof48clinicaldiseasedpigs,29wereconfirmedP
7、RRSVinfectedbyrea-ltimePCR,inwhichonly19wereconfirmedbyconventionalPCR.ThisTaqMan-basedrea-ltimePCRassayisaspecific,sensitiveandquicktoolsuitableforearlyandquickdetectionofPRRSVinclinicallabs.Keywords:Porcinereproductiveandrespiratorysyndromevirus;rea-ltimequantitativePCR;TaqMan-base
8、dprobe豬繁殖與
