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1、_ArchivesArchVirol(1992)125:25-37Vifrology©Springer-Verlag1992PrintedinAustriaRadioactiveandenzymaticclonedcDNAprobesforbovineentericcoronavirusdetectionbymolecularhybridizationJ.Collomb1,C.Finance1,S.AlabouehIandJ.Laporte21LaboratoiredeMicrobiologieMol~cul
2、aire,Facult~desSciencesPharmaceutiquesetBiologiques,Universit~deNancyI,Nancy2StationdeVirologicetd'ImmunologieMol~culaires,INRA,Jouy-en-Josas,FranceAcceptedDecember23,1991Summary.GenomicRNAofF15strainbovineentericcoronavirus(BECV)wasclonedinE.coli.Threeclon
3、es(174,160,PG78),selectedinthecDNAlibrary,includingalargeportionofthenucleocapsid(N),matrix(M)andpeplomeric(S)proteingenes,wereusedasprobesforaslotblothybridizationassay.Twoprobelabellingtechniqueswerecompared,radiolabellingwith32pandenzymaticlabellingthrou
4、ghcovalentlinkagetoperoxidaseandchemilu-minescencedetection.Theradioactiveprobe174detectedaslittleas1to3pgofviralRNA,whilethelesssensitiveenzymaticprobecouldnotrevealmorethan100pgofRNA.Nosignificantdetectionamplificationwasachievedwhenamixtureofthethreeprob
5、eswasused.Probe174allowedspecificidentificationforBECV.NohybridizationwasnoticedeitherwithrotavirusesorevenwithotherantigenicallyunrelatedmembersofthefamilyCoronaviridaesuchastransmissiblegastroenteritisvirus.ThetestprovedvalidfordetectionofBECVinthesuperna
6、tantofinfectedHRT-18cells:genomicRNAcouldbedetectedafterdirectspottingofsamples,butpriornucleicacidextractionafterproteinaseKtreatmentimprovedvirusdetection.BECVdiagnosisinfaecalsamplesusingenzymaticprobewascomparedwithconventionaldiagnosticmethods.Introduc
7、tionThebovineentericcoronavirus(BECV),amemberofthefamilyCoronaviridae,isapleiomorphicenvelopedsphericalparticle(120nmindiameter)surroundedbyacharacteristicfringeof"club-shaped"peplomers.Theviralgenomeisasingle-strandedpolyadenylatedRNAofapproximately20kbinl
8、ength,codingforaY-coterminal"nestedset"ofmRNAsand4majorstructuralproteins:aphosphorylatednucleocapsidproteinN(50k),atransmembranematrixgly-coproteinM(20k),apeplomericglycoproteinS(presentonthevirionas1
