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1、JournalofMedicalVirology29:70-73(1989)DetectionofHumanCoronavirus2293inNasalWashingsUsingRNA:RNAHybridisationS.Myint,S.Siddell,andD.TyrrellInstituteofVirology,UniversityofWiirzburg,FederalRepublicofGermany(S.M.,S.S.);MRCCommonColdUnit,Salisbury,England(D.T.)Amethodisdescribedforthed
2、etectionofhu-zymes.32P-labellednucleotideswerepurchasedfrommancoronavirus229E(HCV229E)innasalAmershamInternational.PromegaiBiotecsuppliedthewashingsusingRNA:RNAfilterhybridisation.plasmidpGEM-1andRQ1DNase.Vanadyl-ribonucle-VolunteerswereinoculatedwithHCV229E,andosidecomplexwaspurcha
3、sedfromBethesdaResearchdailynasalwashingswerecollected.Thesewash-Laboratories.Allotherchemicalsweresuppliedbyingswerethenexaminedforthepresenceofvi-Sigma.ralRNAusingasingle-strandedRNAprobe.Nu-cDNACloningandSubcloningcleicacidhybridisationisshowntobeasensitivetechniqueforthediagnosi
4、sofHCVTheisolationofHCV-specificcDNAcloneswillbe229Einfections.describedindetailelsewhere(Myintetal.,submitted).Briefly,usingamethodbasedonthatofGublerandKEYWORDS:coronavirusdiagnosis,Ribo-Hoffmann[19831,cDNAsweregeneratedfromHCVprobes,nucleicacids2293RNAisolatedfrominfectedC16cells
5、[Phillpotts,19831.OnecDNA,whichcontainedtheentireopenreadingframeofthenucleocapsidgene,wasinsertedINTRODUCTIONintothepolylinkerregionofthe“Riboprobevector,”pGEM-1.Thisplasmid,pSMGF1,haspromotorse-Coronavirusesareagroupofpositive-strandRNAquencesforSP6andT7RNApolymerasesflankingthevi
6、rusesthatcauseawidespectrumofdiseaseinmam-multiplecloningsite,andthussingle-stranded,HCV-malsandbirds[Siddelletal.,19831.Humancoronavi-specificRNAtranscriptscanbegenerated.rusesarethoughttocauseabout15%ofallcommoncolds[Monto,19821andhavealsobeenassociatedwithProbePreparationlowerres
7、piratorytractinfection[Isaacsetal.,1983;RNAprobesweretranscribedandlabelledwith32PMcIntoshetal.,19741.Otherdiseaseassociationshaveusingthefollowingreaction:4p15xtranscriptionbeensuggestedbutarelesswelldocumented[Mac-buffer(0.2MTrisHC1,pH7.5,30mMMgC12,50mMNaughtonandDavies,1981;Riski
8、andHovi,19801.NaC1,
