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1、ELSEV1ERVeterinaryMicrobiology42(1994)65-77DetectionoffelinecoronavirusesincellculturesandinfreshandfixedfelinetissuesusingpolymerasechainreactionXiantangLi*,FredricW.ScottCornellFehneHealthCenterandDepartmentofM~crobtology,ImmunologyandParasitology,CollegeofVetennaryMe&cme,Cor
2、nellUmversgy,Ithaca,NY,14853USAReceived10November1993;accepted1March1994AbstractFelinecoronavirusinfectionsincellculturesandinfreshandfixedfelinetissuesweredetectedusingapolymerasechainreaction(PCR)test.Cellcultureswereinoculatedwithfelineinfectiousperitonitisvirus(FIPV),feline
3、entericcoronavirus(FECV)orshaminoculum.Thetissuesamplesofliver,kidneyandspleenweretakenfromspecific-pathogen-free(SPF)catsthatwereinoculatedintranasallywith103TCIDsoofFIPV79-1146(n=10),FIPVUCD1(n=3)orshaminoculum(n=3),fromclinicalcats(n=43),andfromformalin-fixedarchivedfelineti
4、ssues(n=49),respec-tively.AdditionaltissuesamplesweretakenfromtheFIPV-inoculatedcats(n=6)andwerekeptat4°C,roomtemperatures(20-24°C)and370(2respectivelyfor0,6,12,24,48,72,and96hoursbeforefrozen(-70°C)forPCRtoevaluatetheeffectsoftheambienttemperaturesandpost-mortemintervalsonthet
5、est.Thesampleswerealsofixedin10%neutrallybufferedformalin,95%ethanol,andBouin'ssolutionrespectivelytoevaluatetheeffectsofthefixativesonthetest.PositivePCRresultswereobtainedfromthecellculturesthatwereinoculatedwithFIPVandFECVandfromtheFIPV-inoculatedcats(13/13).NegativePCRresul
6、tswereobtainedfromthesham-inoculatedcellculturesandcats(3/3).Ofthe92clinicalcats,7ofthe8FIP-suspectedcats(87.5%)and51ofthe84non-FIP-suspectedcats(60.7%)wereshowntobevirus-positiveinatleastoneofthetissuesamples.TherewasnosignificantdifferenceinthePCRresultsbetweenthefreshandthef
7、ormalin-fixedtissuesoftheclinicalcats(P>0.05).OftheFIPVinoculatedcats,theviruswasdetectableequallywellinfreshandformalin-,Bouin'ssolution-orethanol-fixedtissues.However,theamountsoftotalRNAextractedfromthefixedtissuesweresignificantlylessthanthosefromfreshtissues(P<0.01).Intiss
8、uesthatwerekeptat4°C,theviruswasdetectableupto96h;atro
