冠状病毒Coronavirusl论文-2016 [Springer Protocols Handbooks] Animal Coronaviruses __ Real-Time RT-PCR Detection of Equine Coronavirus.pdf

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1、Chapter8Real-TimeRT-PCRDetectionofEquineCoronavirusFabienMiszczak,NathalieKin,VincentTesson,andAstridVabretAbstractEquinecoronavirus(ECoV)isarecentlyidentiÞedequinevirus,involvedmainlyinentericinfections.SincetheECoVdiscoveryin1999,onlytworeal-timeRT-PCRshavebeendeve

2、lopedforviralidentiÞca-tion.Inthischapterwedescribeaone-stepreal-timeRT-PCRthathasbeenroutinelyusedinourlabora-toryforECoVdetectionfromfecalandrespiratorysamples.KeywordsECoV,Real-timeRT-PCR,Moleculardetection,Clinicaldiagnosis1IntroductionEquinecoronavirus(ECoV)isaB

3、etacoronavirus-1inthelineageA(betacoronavirusA1)thatwasidentiÞedattheendofthelastcentury[1].ECoVbelongstothegenusBetacoronavirusandiscloselyrelatedtohumancoronavirusOC43(HCoV-OC43),bovinecoronavirus(BCoV),caninerespiratorycoronavirus(CRCoV),bubalinecorona-virus(BuCoV

4、),andporcinehemagglutinatingencephalomyelitisvirus(PHEV).ECoVwasÞrstisolatedinNorthCarolina,USA,fromthefecesofadiarrheicfoalin1999(ECoV-NC99)[2].MultipleECoVoutbreakshaverecentlybeenreportedinJapan[3,4]andintheUSA[5].Majorclinicalsignsobservedwereanorexia,fever,leth-

5、argy,leukopenia,anddiarrhea,andunspeciÞcdiscretesymptomsthatdonotlendtorapiddiagnosis.ECoVwasmainlydetectedinfecalsamplesfromhorsesandlessfrequentlyinrespiratorysecretions[5Ð7].Asmallnumberofanimalswithsignsofencephalopathicdis-easehavealsobeenobservedduringtheseoutb

6、reaks[8].ThecurrentdiagnosisofECoVinfectioncanbeperformedusingvirusisolation,electronmicroscopy,serology[9].Areversetranscrip-tionloop-mediatedisothermalampliÞcation(RT-LAMP),anon-PCR-basednucleicacidampliÞcationassay,hasbeenrecentlydevelopedforthedetectionofECoVinfe

7、calsamples[10].ECoVwasalsoidenti-Þedbymolecularmethodsinfecesandrespiratorysamplesoffoalswithandwithoutentericdisease[2,11,12].Real-timeRT-PCRassaysLeyiWang(ed.),AnimalCoronaviruses,SpringerProtocolsHandbooks,DOI10.1007/978-1-4939-3414-0_8,©SpringerScience+BusinessMe

8、diaNewYork20169394FabienMiszczaketal.canenableapromptidentiÞcationofECoVinrespiratoryandfecalsamplesofhorseswhowereattheearlystageo