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1、BRIEFREPORTS96-wellmicroplates,asystemintendedtoisolatecommonrespira-DETECTIONOFHUMANCORONAVIRUS-NL635toryinfection-relatedviruses:Madin-Darbycaninekidney;Vero;INCHILDRENINJAPANHep-2;humanembryofibroblasts;andHMV-IIcells.SpecimensAkiraSuzuki,MD,*MichikoOkamoto,BM,*AkiraOhmi,*negativefo
2、rvirusisolationwerekeptat80°CuntilthisstudywasOshiWatanabe,MD,*ShigeakiMiyabayashi,MD,PhD,commenced.Thepreviouslyvirus-negativespecimensweresubjectedtoandHidekazuNishimura,MD,PhD*RT-PCRtodetecthumanmetapneumovirus(hMPV)andHCoV-Abstract:HumancoronavirusNL63recentlyfoundintheNether-NL6
3、3asfollows.TotalRNAwasextractedfromeachspecimenlandshasbeendetectedinJapanwithareversetranscription-withtheRNeasykit(Qiagen,Hilden,Germany).Thecomplemen-polymerasechainreactiontechniqueinclinicalspecimensfromtaryDNAwasobtainedbyreversetranscription(RT)reactionusingpediatricpatientswit
4、hrespiratorysymptoms.Of419specimensthatrandomhexamersandMoloneymammaryleukemiavirus-RT(In-werenegativeforcommonrespiratoryviruses,5werepositiveforvitrogen,Carlsbad,CA)asrecommendedbythemanufacturer.ThehumancoronavirusNL63,andthesespecimenswereallcollectedinPCRwasperformedwiththeExTaqk
5、it(TaKaRaBio,Otsu,Japan)thefirst3monthsof2003.andforwardandreversespecificprimers.ForHCoV-NL63,wefollowedthemethodintendedtodetectthe1bgene,describedasKeyWords:humancoronavirus-NL63,humanmetapneumovirus,3diagnosticPCRbyvanderHoeketal,using100ngofprimerrespiratoryillness,JapanrepSZ-1(5-
6、gtgatgcatatgctaattg-3)and100ngofprimerrepSZ-3AcceptedforpublicationJanuary27,2005.(5-ctcttgcaggtataatccta-3).OurPCRconsistedofthefollowingFromthe*VirusResearchCenterandthe†DepartmentofPediatrics,Sendaisteps:95°Cfor2minutes;then35cyclesof94°Cfor1minute,48°CMedicalCenter,Sendai,Japan
7、for1minuteand72°Cfor1minute;then72°Cfor7minutes.WedidReprintsnotavailable.notattemptnestedPCRforthedetectionofthe1bgene.ForhMPV,Copyright©2005byLippincottWilliams&WilkinsweperformednestedPCRusingspecificprimerstoamplifyapartofDOI:10.1097/01.inf.0000168846.71517.ee6theNgeneasdescribedel
8、sewhe
