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1、ArchVirol(1989)104:335-337VirologyArchives©bySpringer-Verlag1989TheuseofnucleicacidhybridizationtodetecthumancoronavirusesBriefReportS.MyintI,S.Siddell1,andD.Tyrrell21InstitutforVirology,UniversityofWiirzburg,Wfirzburg,FederalRepublicofGermany2MRCCommonColdUnit,
2、Salisbury,EnglandAcceptedJanuary6,1989Summary.WehaveappliedanRNA:RNAhybridizationtestforthedetectionofhumancoronavirus229E.ThistestisundergoingfurtherdevelopmentbutalreadyallowsadiagnosisofHCV229Einfectionwithin48hours.Humancoronavirusesarepositive-strandRNAviru
3、sesthatarethoughttocauseabout20%ofallcommoncolds.Theyhavealsobeenassociatedwithotherdiseases[1]butonlytheassociationwithrespiratorytractinfectionissubstantiated[2].Coronaviruseshaveanunusualreplicationstrategywhichinvolvesa3'coterminalsetofsubgenomicmRNAs.Onlyth
4、e5'proximalregionofeachmRNAgenerallycodesforaprotein.InthecaseofhumancoronavirusesthesmallestofthesesubgenomicmRNAscodesforthenucleocapsidprotein.Wehaveusedanoligo-dTprimerandamethodbasedonthatofGublerandHofmann[3]tocreatealibraryofhumancoronavirus(HCV)229EcDNAc
5、lones.Fromthislibrary,cDNArepresentingacompletecopyofthenucleo-capsidgenehasbeenidentifiedandsubclonedintothe'riboprobe'vectorpGEM-1.ThisvectorhasapromotersitefortheattachmentofT7RNApolymerase,usingwhich32p-labelledsinglestrandedRNAtransciptscanbegenerated.These
6、transciptshavebeenshowntobemoresensitivethannick-translateddouble-strandedDNAprobesinthedetectionofHCV229ERNA[Myintetal.,inprep.].TheirspecificityhasbeenexaminedbyhybridizationtoaseriesoffiltersontowhichhadbeenboundRNAfrom42commoncoldpathogens(HCV229E,HCVOC43,HC
7、V229E/Killick,4parainfuenza,4in-fluenzaand31rhinoviruses),onlytheHCV229Egroupweredetected(unpub-lisheddata).This"riboprobe"hasnowbeensuccessfullyappliedtothedetectionofHCV229Einnasalwashingscollectedfromvolunteersinfectedwiththevirus336S.Myintetal.Fig.1.Hybridiz
8、ationof32p-labelledRNAprobetonasalwashingsfromHCV229Einoculatedvolunteers.(Seetextfordetails)inacontrolledtrial.Ofeightvolunteersinoculatedintra-nasaltywithHCV229Efou
