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1、浙江农业学报ActaAgriculturaeZhejiangensis20(5):328~332,2008猪繁殖与呼吸综合征病毒荧光定量PCR检测方法的建立1,*1,21112徐辉,方立,陈伟杰,赵灵燕,倪柏锋,方维焕(1浙江省畜牧兽医局,浙江杭州310020;2浙江大学动物预防医学研究所,浙江省重点实验室,浙江杭州310029)摘要:针对猪繁殖与呼吸综合征病毒(Porcinereproductiveandrespiratorysyndromevirus,PRRSV)N基因保守序列设计引物与TaqMan探针,在建立常规PCR的基础上,设计并优化了TaqMa
2、n荧光定量PCR方法用于PRRSV核酸的检测。应用新建立的荧光定量PCR方法检测其它主要猪病病原,无任何非特异性反应;针对PRRSV1阳性克隆质粒的检测灵敏度可以达到1.210copy/ml,比常规PCR高100倍;通过对48份临床疑似样本的检测表明有29份样本为PRRSV阳性,与巢式PCR检测结果一致,而常规PCR只能检出其中19份阳性样本,灵敏度优于常规PCR方法。结果表明,试验建立的PRRSVTaqMan荧光定量PCR方法具有良好的特异性、敏感性和重复性,可为PRRSV临床诊断提供一个更为有效的方法。关键词:猪繁殖与呼吸综合征病毒;荧光定量PCR;Taq
3、Man探针中图分类号:S828文献标识码:A文章编号: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猪繁殖与
