冠状病毒Coronavirusl论文-2016 [Springer Protocols Handbooks] Animal Coronaviruses __ Detection of Bovine Coronavirus by Conventional Reverse Tran.pdf

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1、Chapter9DetectionofBovineCoronavirusbyConventionalReverseTranscriptionPolymeraseChainReactionAmauriAlcindoAlfieri,AliceFernandesAlfieri,andElisabeteTakiuchiAbstractBovinecoronavirus(BCoV)isaneconomicallysigniÞcantcauseofentericandrespiratorydiseasesincattlethrou

2、ghouttheworld.BCoVisaknowncauseofneonatalcalfdiarrhea,winterdysenteryinadultcattle,andrespiratorydisordersincattleofallages.Inthischapter,wedescribeasimpleandefÞcientprotocolfortotalnucleicacidsextractiontobeusedinconventionalRT-PCRassay.Thisisatechniqueusedro

3、utinelyinourvirologylaboratorytodetectBCoVfromstoolandnasopharyngealsamplesofcattle.KeywordsBovinecoronavirus,RT-PCR,Clinicaldiagnosis,Stoolsamples,Nasopharyngealsamples1IntroductionBovinecoronavirus(BCoV)isamemberoftheorderNidovirales,Coronaviridaefamily,whic

4、hwasrecentlyclassiÞedasmemberofthespecieBetacoronavirus1inthegenusBetacoronavirus[1].BCoVarefrequentlycirculatingincattlefarmsworldwide,causingbothentericandrespiratorydiseaseincalvesandadultcattle[2].Becausetherespiratoryandentericdisordersaresimilartotheothe

5、rinfectiousdiseases,aspeciÞctestisneededforacon-clusivediagnosisofBCoVinfection.BesidessensitivetestsarerequiredtodetectBCoVespeciallyatearlyorlatestagesofdiseasewhentheyhavelowlevelsofviralshedding.ThecurrentmethodsusedforthediagnosisofBCoVconsistmostlyofthed

6、etectionofviralRNAbyconventional(RT-PCR)andreal-timereversetranscriptionpolymerasechainreaction(qRT-PCR)[3Ð6].Recently,isothermalnucleicacidampliÞcationtechniques,suchasrecombinasepolymeraseampliÞcation(RPA)assayandreversetranscriptionloop-mediatedisothermalam

7、pliÞ-cation(RT-LAMP),havebeendevelopedforrapiddetectionofBCoV[7,8].However,therearestillfewreportsevaluatingLeyiWang(ed.),AnimalCoronaviruses,SpringerProtocolsHandbooks,DOI10.1007/978-1-4939-3414-0_9,©SpringerScience+BusinessMediaNewYork2016101102AmauriAlcindo

8、Alfierietal.thesenewassaysinlarge-scaleepidemiologicalstudiesofBCoVinfections.Inthischapter,wedescribeasensitiveandspeciÞcconven-tionalRT-PCRassaythathasbeensuccessfullyappliedfordi