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1、fluenzavirustypes1,2and3andinfluenzavirusesAandBBriefReportsintheNPAandMEFsamples.Thesampleshadbeenana-lyzedforhumanrhinovirusbyapreviouslypublished,com-10binedisolation-RT-PCRmethod.AsystematicreportonoccurrenceoftheseviruseswillbeRESPIRATORYCORONAVIR
2、USINFECTIONSINpreparedseparately.CHILDRENYOUNGERTHANTWOYEARSOFAGEHCoVRT-PCRandhybridization.ExtractionofviralThehumancoronaviruses(HCoV)comprisemanyanti-RNAfromNPAandMEFsampleswasperformedwithagenicvariants,withthetwoserogroupsrepresentedbycommercialRN
3、Aisolationprocedure(RNeasy,QiagenstrainsOC43and229Ebeingthemostcommonones.HCoVGmbH,Hilden,Germany).RT-PCRforHCoVRNAwasinfectionshavebeenassociatedwithcommoncoldsinchil-carriedoutbypreviouslypublishedmethodswithminor8,10drenandadultsandwithwheezinginyou
4、ngchildrenwithmodifications.Pairsofprimerscomplementarytothe1,2HCoVnucleocapsidproteingeneforbothHCoVOC43andrecurrentrespiratoryinfections.ReinfectionswithHCoVarecommon,andthevirusmaybecarriedforextended229E,50pmolofeach,wereusedinthesamereaction.Atthe
5、23reversetranscriptionstepthevirus-specificoligonucleotideperiodsintheupperrespiratorytract.PitkaÈrantaetalrecentlyfoundHCoV,usingthereversetranscriptasepoly-primerswere59-GCAAGAATGGGGAACTGTGG(OC43)andmerasechainreaction(RT-PCR)technique,in17%ofthe59-G
6、ACTATCAAACAGCATAGCAGC(229E).InthePCRthemiddleearfluid(MEF)/nasopharyngealaspirates(NPA)in59-biotinylatedforwardprimerswere59-AGGAAGGTCT-childrenwithacuteotitismedia(AOM).InFinland40%ofGCTCCTAATTC(OC43)and59-GGTACTCCTAAGCCT-childrenwerereportedtohaveant
7、ibodiesagainstthestrainTCTCG(229E).ReversetranscriptionwascarriedoutinaOC43bytheageof2yearsandpracticallyallofthemhadfinalvolumeof40mlincluding5mlofRNA.Themixtures4,5wereincubatedfor60minat37°Candthenheatedfor10minantibodiesbytheageof6years.However,noe
8、xtensivestudiestodefinebettertheroleofHCoVinrespiratoryat65°C.PCRwasperformedin96-wellplatesinafinalinfectionshavebeenundertaken.volumeof100mlwith5mlofcomplementaryDNA.Forty8AlthoughHCoVinfectionsarecommon,routinediagnosticcyclesofPCRwe
