冠状病毒Coronavirusl论文-2016 [Springer Protocols Handbooks] Animal Coronaviruses __ Real-Time Reverse Transcription Polymerase Chain Reaction fo.pdf

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1、Chapter10Real-TimeReverseTranscriptionPolymeraseChainReactionforRapidDetectionofTransmissibleGastroenteritisVirusRameshVemulapalliAbstractTransmissiblegastroenteritis(TGE)isahighlycontagiousdiseaseofpigscausedbytheTGEvirus(TGEV).Rapiddetectionofthevi

2、rusintheaffectedpigsfecesiscriticalforcontrollingthediseaseoutbreaks.Thereal-timeRT-PCRassaydescribedinthischaptercanquicklydetectthepresenceofTGEVinfecalsampleswithhighsensitivityandspecificity.KeywordsTGEvirus,Real-timeRT-PCR,Feces,Pigs1Introduction

3、Transmissiblegastroenteritis(TGE)isahighlycontagious,acuteviraldiseaseofpigs[1].TGEcanaffectpigsofallages,butthediseaseseverityandmortalityratearehighinpigletsunder2weeksofage.ThecausativeagentisTGEvirus(TGEV),acoronavirusthatprimarilyinfectsandrepli

4、catesintheepithelialcellsofpigintestines.Affectedanimalsshedthevirusintheirfeces.Thedis-easetransmissionoccursprimarilyviafecal-oralroute[2].Duringanoutbreak,rapiddetectionofTGEVinfecesisveryusefulforimplementingthediseasemanagementpracticesinatimely

5、man-ner.AnyTGEV-specificdiagnosticassaymustbeabletodifferen-tiateitfromporcinerespiratorycoronavirus(PRCV),anaturalmutantofTGEVwithtruncatedspikeproteinandalteredcelltropismtowardsrespiratoryepithelialcells[3].PRCVmostlycausesmildorsubclinicalrespirat

6、orydisease.However,insomePRCV-infectedpigs,theviruscanbeshedinthefeces[4].Real-timePCR-basedassaysarewellsuitedforrapid,specific,andsensi-tivedetectionofvirusessuchasTGEV.Thereal-timeRT-PCRassaydescribedhereisbasedonamplificationofaconservedregionofthe

7、spikeproteingeneofTGEVstrainsanddetectionofLeyiWang(ed.),AnimalCoronaviruses,SpringerProtocolsHandbooks,DOI10.1007/978-1-4939-3414-0_10,©SpringerScience+BusinessMediaNewYork2016115116RameshVemulapallitheamplifiedproductsusingaTaqManprobe[5].Theassay,a

8、longwiththeRNAextractionmethoddescribedhere,canbeestablishedinanymoleculardiagnosticlaboratoryfordetectionofTGEVinpigfecalsamples[68].2Materials2.1RNAExtraction1.TRIzolLSReagent(Invitrogen).2.RNeasyMiniKit(Qiagen).3.Chloroform.4.Ethanol,70and96100%.5