冠状病毒Coronavirusl论文-2008 [Methods in Molecular Biology] SARS- and Other Coronaviruses Volume 454 __ A Pancoronavirus RT-PCR Assay for Detect.pdf

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1、1APancoronavirusRT-PCRAssayforDetectionofAllKnownCoronavirusesLeenVijgen,ElienMoës,ElsKeyaerts,SandraLi,andMarcVanRanstAbstractTherecentdiscoveriesofnovelhumancoronaviruses,includingthecoronaviruscausingSARS,andthepreviouslyunrecognizedhumancoronavirusesH

2、CoV-NL63andHCoV-HKU1,indicatethatthefamilyCoronaviridaeharborsmoremembersthanwaspreviouslyassumed.Allhumancoronavirusescharacterizedatpresentareassociatedwithrespira-toryillnesses,rangingfrommildcommoncoldstomoreseverelowerrespiratorytractinfections.Since

3、theetiologyofarelativelylargepercentageofrespiratorytractdiseasesremainsunidentified,itispossiblethatforacertainnumberoftheseillnesses,ayetunknownviralcausativeagentmaybefound.Screeningforthepresenceofnovelcoro-navirusesrequirestheuseofamethodthatcandetec

4、tallcoronavirusesknownatpresent.Inthischapter,wedescribeapancoronavirusdegenerateprimer-basedmethodthatallowsthedetectionofallknownandpossiblyunknowncoronavirusesbyRT-PCRamplificationandsequencingofa251-bpfragmentofthecoronaviruspolymerasegene.Keywords:co

5、ronavirus;pancoronavirusRT-PCR;degenerateprimers;sequencing;polymerasegene1.IntroductionAtpresent,viralcultureisthegoldstandardforlaboratorydiagnosisofrespiratoryinfections.Sincecoronavirusesareverydifficulttogrowincellculture,accurateandsensitivediagnose

6、sarenotfeasiblebythistechnique.Toovercomethelackofsensitivityandtoobtainrapiddiagnosticresults,moresensitivemolecularmethodsforthedetectionofhumancoronaviruses(HCoVs)havebeendeveloped,includingreverse-transcriptasepolymerasechainreactionFrom:MethodsinMole

7、cularBiology,vol.454:SARS-andOtherCoronaviruses,Editedby:D.Cavanagh,DOI:10.1007/978-1-59745-181-91,CHumanaPress,NewYork,NY34Vijgenetal.(RT-PCR),nestedRT-PCR,andrecentlyreal-timeRT-PCR(1).Nevertheless,HCoVsarenotoftendiagnosedinclinicallaboratories,althou

8、ghtheSARSepi-demicandtheidentificationofHCoV-NL63drewattentiontotheclinicalrele-vanceofHCoVs.MultiplexRT-PCRsforthedetectionofcommonrespiratoryvirusesinclinicalspecimenshavenowbeensupplementedwithprimerpairsforampli