冠状病毒Coronavirusl论文-2007 SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronavirus.pdf

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1、ArchVirol(2007)152:4158DOI10.1007/s00705-006-0840-xPrintedinTheNetherlandsSYBRGreenreal-timereversetranscription-polymerasechainreactionassayforthegenericdetectionofcoronaviruses∗S.Escutenaire1,N.Mohamed2,M.Isaksson1,P.Thoren´1,B.Klingeborn1,S.Belak´1,3,M.Berg3,andJ.Blomberg21DepartmentofVirolo

2、gy,NationalVeterinaryInstitute,Uppsala,Sweden2DepartmentofMedicalSciences,SectionofClinicalVirology,AcademicHospital,UppsalaUniversity,Uppsala,Sweden3DepartmentofBiomedicalSciencesandVeterinaryPublicHealth,SectionofParasitologyandVirology,SwedishUniversityofAgriculturalSciences,Uppsala,SwedenRe

3、ceivedMay4,2006;acceptedJuly12,2006PublishedonlineAugust28,2006cSpringer-Verlag2006Summary.Coronavirusesareetiologicagentsofrespiratoryandentericdiseasesinhumansandinanimals.Inthisstudy,aone-stepreal-timereversetranscription-polymerasechainreaction(RT-PCR)assaybasedonSYBRGreenchemistryanddegen

4、erateprimerswasdevelopedforthegenericdetectionofcoronaviruses.Theprimers,designedintheopenreadingframe1b,enabledthedetectionof32animalcoronavirusesincludingstrainsofcaninecoronavirus,felinecoronavirus,transmissiblegastroenteritisvirus(TGEV),bovinecoronavirus(BCoV),murinehepatitisvirus(MHV)andin

5、fectiousbronchitisvirus(IBV).Aspecificamplifi-cationwasalsoobservedwiththehumancoronaviruses(HCoV)HCoV-NL63,HCoV-OC43,HCoV-229Eandsevereacuterespiratorysyndromecoronavirus(SARS-CoV).Thereal-timeRT-PCRdetecteddownto10cRNAcopiesfromTGEV,BCoV,SARS-CoVandIBV.Inaddition,theassayexhibitedahighsen-sitiv

6、ityandspecificityonclinicalsamplesfromdifferentanimalspecies.Thedevelopedassayrepresentsapotentialtoolforlaboratorydiagnosticsandfordetectingstilluncharacterizedcoronaviruses.IntroductionCoronavirusesareenvelopedpositivesingle-strandedRNAviruses,membersoftheorderNidovirales[8].Theirgenomeis2731k

7、binlengthandiscomposedinits5-proximaltwo-thirdsoftwolargeopenreadingframes(ORFs),ORF1aandORF1b,encodingthereplicasecomplex[1].Genesencodingthestructuralpro-∗ThestudywasperformedattheDepartmentofVirology,SVA,Uppsala,Sweden.42S.Esc