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1、JournalofVirologicalMethods,29(1990)243-256243ElsevierVIRMET01051PolymerasechainreactionforprobesynthesisandfordirectamplificationindetectionofbovinecoronavirusArnoldVerbeekandPeterTijssenCentrederechercheenmCdicinecomparJe,InstitutArmand-Frappier,Uni
2、versite’duQuebec,Laval-des-Rapides,Canada(Accepted10May1990)SummaryThepolymerasechainreaction(PCR)wasusedtosynthesizedsandssprobesforthedetectionofbovinecoronavirus(BCV)usingrecombinantplasmidsastemplatemolecules.Thedsprobesdetectedaminimumofabout2x10
3、viralgenomesafterexposurefor1h,adetectionlimitsimilartonick-translatedprobesafterexposureofthefilmsfor60h.Morethan8hexposuretoblotsprobedwiththesedsprobesresultedincompletedarkeningofthefilm.Thessprobes,synthesizedbyasymmetricPCRonlinearizedplasmids,p
4、ermittedthedetectionof5x104genomes,whichequalledthecapacityofrandom-primedprobes.ProlongedexposuredidnotincreasethebackgroundasincaseofdsPCR-probedblots.Probes,synthesizedbyasymmetricPCRandrandom-primingwerelabeledtosimilarspecificactivitiesandwerebet
5、terintermsofsensitivityanddetectabilityasopposedtonick-translatedprobes.However,thespecificityofdetectionwithssprobesastorandomprimedprobeswasincreasedfurther.About10viralgenomes,afterfragment-specificamplificationbyPCR,weredetectedbyagarose-gelanalys
6、is.PCR-probesynthesiswassimple,highlyreproducible,andallowedthesynthesisofprobesforspecificfragments.Bovinecoronavirus;Polymerasechainreaction;Probelabeling;DetectionCorrespondenceto:P.Tijssen,Centrederechercheenmedicinecornparke,InstitutArmand-Frappi
7、er,Universit6duQuCbec,531,boul.desPrairies,Laval-des-Rapides,QC,CanadaH7VlB7.0166-0934/90/$03.5001990ElsevierSciencePublishersB.V.(BiomedicalDivision)244IntroductionNeonataldiarrheainnewborncalvesisasyndromeofgreatetiologicalcomplex-ity.Themajorinfect
8、iousorganismsassociatedwiththediseasearecoronaviruses,rotaviruses,andenterotoxigenicE.coli(Mebusetal.,1973;Bridgeretal.,1978;Saifetal.,1986),whileseveralconditionalfactorsplayaninfluentialroleindis-easedevelopment.Numeroustestsforthedetectiono
