冠状病毒Coronavirusl论文-2000 Development of a Reverse Transcription-Nested Polymerase Chain Reaction Assay for Differential Diagnosis of Transmi.pdf

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1、Briefcommunications385JVetDiagnInvest12:385±388(2000)Developmentofareversetranscription±nestedpolymerasechainreactionassayfordifferentialdiagnosisoftransmissiblegastroenteritisvirusandporcinerespiratorycoronavirusfromfecesandnasalswabsofinfectedpigsLomiKim,Kyeong-OkChang,KarolSestak

2、,AnilParwani,LindaJ.SaifAbstract.Transmissiblegastroenteritisvirus(TGEV),acoronavirus,replicatesinintestinalenterocytesandcausesdiarrheainyoungpigs.Porcinerespiratorycoronavirus(PRCV),aspike(S)genenaturaldeletionmutantofTGEV,hasarespiratorytissuetropismandcausesmildorsubclinicalresp

3、iratoryinfections.Conventionalantigen-baseddiagnostictestsfailtodifferentiateTGEVandPRCV,andablockingELISAtesttoserologicallydifferentiateTGEV/PRCV-infectedpigsisconductedonconvalescentserumretrospectivelyafterdiseaseout-breaks.Areversetranscription(RT)-nestedpolymerasechainreaction

4、(PCR)withprimerstargetedtotheSgenedeletionregiontodifferentiateTGEV/PRCVwasdeveloped.Thespeci®cityoftheRT-nestedPCRwascon®rmedwithreferenceandrecent®eldstrainsofTGEV/PRCV,anditssensitivitywasanalyzedbytestingnasalandfecalsamplescollectedfrompigsatvariousdayspostinoculation(DPI)withT

5、GEVorPRCV.Speci®cPCRproductsforTGEV/PRCVweredetectedonlywiththehomologousreferenceor®eldcoronavirusesandfor10±14DPIofpigswithTGEV(feces)orPRCV(nasalsamples).TheRT-nestedPCRassaywasmoresensitivethanantigen-basedassaysonthebasisofdurationofvirusdetectioninexperimentallyinfectedpigsand

6、wasdirectlyapplicabletonasalaswellasfecalspecimensfromthe®eld.Transmissiblegastroenteritisvirus(TGEV)isamemberherdtestedonlyPRCVpositive,whereasotherswereTGEVoftheCoronaviridaefamilyandisenvelopedwithaposi-positive(inconsistentherdimmunestatus).Thesenewtive-strandedRNAgenome.3,7Porc

7、inerespiratorycoronavi-TGEVstrainsmayrepresentnaturallyoccurringrecombi-rus(PRCV)representsanaturaldeletionmutantofTGEVnantswithreducedvirulencebetweenTGEVandPRCVthatappearedin1983±1984inEuropeandin1988inthestrains,orthepresenceofPRCVantibodiesintheseherdsUS.3Coronaviruseshave3major

8、structuralproteins: