pcr检测水痘病毒

pcr水痘病毒 JournalofVirologicalMethods113(2003) 113–116 Shortcommunication Areal-timePCRassayforthedetectionofvaricella-zostervirusDNA anddifferentiationofvaccine,wild-typeandcontrolstrains GrahamA.Tipples∗,DavidSafronetz,MichaelGray NationalMicrobiologyLaboratory,HealthCanada,1015ArlingtonStreet,Winnipeg,Man.,CanadaR3E3R2 Received20May2003;receivedinrevisedform23July2003;accepted24July2003 Abstract Varicella-zostervaccineisaliveattenuatedvirus.Itis,therefore,necessarytohaveatesttodifferentiatevaccinefromwild-typevaricella-zostervirus(VZV)strainsfortheinvestigationofvaricellaorzoster-likerashillnessinindividualsvaccinatedpreviously.Inaddition,itisnecessarytohavearapidVZVassayforuseinthecontextofsmallpoxbioterrorismlaboratorytesting.Usingspecificprimersandhybridizationprobes,arapidmethodtodifferentiatevaccinestrainVZVfromwild-typeVZVwasdevelopedbasedonthepresenceorabsenceofaPstIrestrictionsitewithinopenreadingframe(ORF)38.UsingthisORF38assayinconjunctionwithasimilarpreviouslydescribedORF62assayallowsforfurtherdifferentiationofvaccinestrain,wild-typeandalaboratorycontrolstrain(Ellen)VZV.ThisisaccomplishedbecauseEllenVZVissimilartowild-typeVZVwithrespecttotheORF38assaybutissimilartovaccinestrainVZVwithrespecttotheORF62assay.ThehybridizationprobesforeachORFarelabeledwithdifferentfluorescenttagsthusallowingbothassaystoberunsimultaneouslyinasingletube.BothassaysdemonstrateahighdegreeofspecificityforVZVandcanreliablydetectbetween10and100copiesofVZVDNA.Thus,thereal-timepolymerasechainreaction(PCR)assayforVZVdescribedbelowprovidesarapidassayallowingthesimultaneousdifferentiationofvaccine,wild-typeandlaboratorycontrolstrainsofVZV.©2003ElsevierB.V.Allrightsreserved. Keywords:Chickenpox;Shingles;LightCycler Primaryinfectionwithvaricella-zostervirus(VZV)causeschickenpox(varicella),acommonchildhoodillnesscharacterizedbyageneralizedvesicularrash.Asatypicalherpesvirus,VZVestablisheslatencyinthehostsensorynerveganglia,andcanreactivateatalatertimetocauseshingles(zoster)inindividualswithwaningimmunity.Al-thoughchickenpoxandshinglesarerarelyfatal,seriousandpainfulcomplicationsareknowntooccur.Inadultsandimmunocompromisedindividualsvaricellaismoresevereandmayresultinvaricellapneumoniaordisseminateddis-ease.Duringpregnancy,varicellacanresultincongenitalmalformationsinthefetusordisseminatedvaricellaintheneonatedependingonthetimeofinfection. ThereisaliveattenuatedVZVvaccinewhichwasde-velopedinJapaninthemid-1970s(TakahashiandPlotkin2000).VZVvaccinewaslicensedforuseintheUSin1995(CentersforDiseaseControl,1996),andinCanadain1998 Correspondingauthor.Tel.:+1-204-789-6080;fax:+1-204-789-5009. E-mailaddress:grahamtipples@hc-sc.gc.ca(G.A.Tipples). 0166-0934/$–seefrontmatter©2003ElsevierB.V.Allrightsreserved.doi:10.1016/S0166-0934(03)00229-5 ∗ (HealthCanada,1999).AstheVZVvaccineisalivevirus,itcanestablishlatencyinthehost.Mildchickenpoxhasbeenshowntobeararevaccineassociatedadverseevent,andthevaccinestrainhasalsobeenshowntoreactivatetocausemildshingles(Liangetal.,1998;White,1996).Inaddition,break-thoughwild-typeVZVinfectionshavebeenshowntooccuroccasionallyinindividualsvaccinatedpreviously(White,1997;Gailetal.,2002).Itis,therefore,necessarytomonitorallVZVinfectionsinpreviouslyimmunizedin-dividualstodeterminewhethertheinfectionistheresultofwild-typeorvaccinestrainvirus. Ingeneral,chickenpoxiseasilydiagnosedclinically.However,laboratoryconfirmationofVZVinfectionsareusefulfordifferentiationfromothervesicularrashillnessessuchasherpessimplex,forthedifferentiationofwild-typeandvaccinestrainsinthelaboratoryinvestigationofchick-enpoxorshinglesinpreviouslyimmunizedindividuals,andnowinanerawithbioterrorismthreats,smallpox.Althoughthelatterinstanceishopefullyarareifnotnon-existentsit-uation,itisstillnecessaryforrapiddiagnosticprocedurestobeinplacetorespondtosuchasituation.Vesicularfluid 114G.A.Tipplesetal./JournalofVirologicalMethods113(2003)113–116 fromVZVlesionscontaininfectiousvirusandVZVcanbeconfirmedbyvirusisolation,directfluorescenceassayus-ingmonoclonalantibodies,orbypolymerasechainreaction(PCR).AsinallPCRdiagnostictests,itisimportanttobeabletoclearlydifferentiatethePCRproductsoftheposi-tivecontrolfromtheclinicalspecimen.Inaddition,weareinterestedindifferentiatingvaccinefromwild-typeVZVstrains.Thereal-timePCRassaywedescribebelowisarapidtestwhichallowsforthedifferentiationofwild-type,vaccineandcontrolVZVstrains. Numerousstudieshavebeenpublisheddescribingdif-ferencesbetweendifferentVZVstrains,inparticulardif-ferencesbetweenthevaccineandwild-typestrains(Argawetal.,2000;HawramiandBreuer,1997;Martinetal.,1982;Shirakietal.,1991).ThePstIsiteinopenreadingframe(ORF)38ofVZVhasbeenshowntobeabsentintheOkavaccinestrainbutpresentinwild-typestrains,withtheex-ceptionofsomeminorJapanesewild-typestrainswhicharePstInegative(LaRussaetal.,1992).ASmaIsiteinORF62hasbeenshowntobeabsentinallwild-typestrains,andpresentintheOkavaccinestrain,andthusbeausefulmarkerfordifferentiatingwild-typefromvaccinestraininfections(Loparevetal.,2000a).Loparevetal.(2000b)havede-scribedareal-timePCRassaybasedonusingthisrestrictionsitetodifferentiaterapidlywild-typeandvaccinestrains.Wehaveextendedthisworktoincludeasimilarreal-timePCRassayforthePstIsiteinORF38.WehavedeterminedthattheEllenVZVstrainisvaccinestrain-likewithrespecttoORF62andwild-type-likewithrespecttoORF38.ByusingtheEllenstrainasourPCRpositivecontrol,andrun-ningtheLoparevORF62real-timePCRinadditiontotheORF38real-timePCR,weareabletodifferentiaterapidlywild-type,vaccinestrainandpositivecontrolVZVstrains.Forwardandreverseprimersweredesignedtoamplifya347basepair(bp)regionofVZVORF38.HybridizationprobeswerethendesignedtobindoverthePstIsitewithinthe347bpampliconallowingfordifferentiationofvaccinestrainVZVfromwild-type.PreviouslydescribedprimersandprobesforORF62werealsousedtoamplifyanddiffer-entiateVZVvaccinestrainfromwild-type(Loparevetal.,2000b).SeeTable1forprimerandprobesequences. Table1 OligonucleotidesequencesforPCRprimersandprobesPrimer/probeSequence(5󰀃to3󰀃) ORF38CGGGTGAACCGTATTCTGAGORF38rTTGAACAATCACGAACCGTT ORF38LCLC-Red640-ACGATATATACCGCAGTTGTTGCGORF38FLGACTTGAAGATGAACTTAATGAAGCCCGTG-FLORF62AACTCGCTGGCCCAAAGGTGORF62rGTGTCCGCTTTGAACGCCCG ORF62LCLC-Red705-AGGTGGCCCAGGGATGGAORF62FLGTTGCTGGTGTTGGACGCGGTGGCCCT-FLBG1CAACTTCATCCACGTTCACCBG2 GAAGAGCCAAGGACAGGTAC FL:fluoresceinlabel. Real-timePCRwascarriedoutontheLightCycler(RocheAppliedSciences,Laval,Que.).Eachreactioncontained2␮lmastermix(FastStartDNAMasterHybridizationProbes,RocheAppliedSciences,Laval,Que.),3mMMgCl2,1␮Moftheforwardandreverseprimer,0.2␮Mofthefluoresceinhybridizationprobe(FLprobe,TIBMOLBIOL,Adelphia,NJ),0.4␮MoftheLC-Red640or705hybridizationprobe(LCprobe,TIBMOLBIOL,Adelphia,NJ),2␮ltemplateVZVDNA.Waterwasaddedtobringthetotalvolumeto20␮l.Followinganinitial10minincubationat95◦C,sam-pleswereamplified◦with45cyclesof95◦Cfor3s,62◦Cfor7s,72Cfor20swithfluorescenceresonanceenergytransfer(FRET)measuredaftereachannealingstep.Afinalextensionstepat72◦Cfor40swasdone. DifferentiationoftheVZVisolateswasachievedthroughmeltingcurveanalysis(Fig.1).Samplesweredenaturedat95◦◦Cfor2minandequilibratedto45◦Cfor40s.From45C,thetemperaturewasslowlyraised(0.3◦C/s)to80◦C,whiletheLightCyclercontinuouslymonitoredtheFRETforeachsample.WiththeORF38primersandprobeswild-typeandEllenVZVhadameltingpeakat60.5◦CwhilevaccinestrainVZVmeltedat67.5◦C.FortheORF62primersandprobeswild-typeVZVhadameltingpeakof69.5◦◦C,whileEllenandvaccinestrainVZVmeltedat61.7C. Thespecificityoftheprimerswasassessedbyrunningsamplespositiveforgenomicmaterialfromotherher-pesvirusesandpotentialrashcausingagents.Thesesamplesincludedallofthehumanherpesviruses(herpessimplexviruses1and2,Epstein–Barrvirus,cytomegalovirus,hu-manherpesviruses6A,6B,7and8),threesimianalpha-herpesviruses(Cercopithecineherpesvirusestype1(CeHV-1,MonkeyBVirus)type2(CeHV-2,SimianAgent-8,SA-8)andtype16(CeHV-16,herpesviruspapio2,HVP-2)),camelpox,anechovirus(E9)isolate,WestNilevirus,denguevirus(serotype2),measlesvirus,rubellavirus,enterovirusEV71,coxsackievirusesA-16andB-4,parvovirusB-19,andthebacteriumLeptospira.BothsetsofprimersprovedtobehighlyspecificwithVZVDNAbeingtheonlyagentamplifiablewitheitherset. Ellen(ATCC#VR-1367),Oka(Varivax,MerckFrost,Kirkland,Que.)andawild-typeVZVisolateswereculturedinMeWocells(agiftfromDr.CharlesGrose,UniversityofIowa)inDMEM(Gibco,Burlington,Ont.)supplementedwith5%FBS,1%non-essentialaminoacids,and1%sodiumpyruvateandincubatedat37◦Cforapproximately3–5days.TheVZVnucleocapsidswereisolatedfrominfectedMeWocellsaspreviouslydescribed(Safronetzetal.,2003).DNAwasextractedfromthenucleocapsidpreparationusingstandardphenol–chloroformmethods.ToensurethepurityoftheviralDNA,PCRwasperformedwithprimersspecificforhumanbeta-globincellularhousekeepinggenes(BG1or2,seeTable1forsequences)aspreviouslydescribed(Safronetzetal.,2003).TheabsorbanceofthepureviralDNAwasmeasuredat260nmandtheviralgenomecopynumberdetermined. G.A.Tipplesetal./JournalofVirologicalMethods113(2003)113–116115 Fluorescence [–d(F2/F1)/dT] 0.0(A) 5060 Tm (˚C) 7080 Fluorescence [–d(F3/F1)/dT] 0.016 0.008 0.0 (B) 5060 Tm (˚C) 7080 ) Fig.1.ORF38(A)andORF62(B)genotypingofVZVbymeltingcurveanalysisontheLightCycler.()Vaccinestrain(Oka)VZV;(wild-typeVZV;()EllenstrainVZV;(—)notemplatecontrol.MeltingtemperaturesforeachVZVstrainareindicatedabovewitharrows. ThesensitivityoftheORF38and62primersandprobeswasassessedbyrunning10-foldserialdilutionsofknowngenomicequivalentsofpurifiedvaccineandwild-typeVZVDNA.Bothsetsofprimersandprobesdetectedreproduciblybetween10and100VZVgenomiccopies. AlthoughthesamplesusedinthisstudywereVZViso-lates,theassayissensitive(10–100genomiccopies)andthusshouldproveusefulfordirectamplificationfromclini-calspecimens(vesicularfluid/swabs).Inaddition,manyoftheclinicalvirologylabsinCanada,fromwhereourrefer-encelabreceivessamplesfortesting,dovirusisolationforVZV.Thus,thisassaywasdevelopedandassessedinourlaboratoryusingclinicalVZVisolates. Atotalof20samples(18clinicalisolate,Ellenstrain,andvaccinestrain)wereblindlytestedontheLightCyclerusingtheORF38and62assaysdescribedaboveandcom-paredwithrestrictionfragmentlengthpolymorphismresultsasoriginallydescribed(LaRussaetal.,1992;Loparevetal.,2000a).The18clinicalisolatesincluded14wild-typesam-ples,includingVZV-BC(Tipplesetal.,2002),andfoursamplesspikedwithOkaDNA.All18samplesweregeno-typedcorrectlyaswild-typeorvaccinestrainsbytheORF38and62assays.Also,theEllencontrolDNAwaseasilydifferentiatedfromthesamples. TheLCprobesusedintheORF38and62assaysarela-beledwithdifferentfluorescenttags(ORF38usesLC-Red640whileORF62usesLC-Red705).ItwasfoundthataftercalibratingtheLightCyclerinstrumentusingacolorcom-pensationkit(RocheAppliedSciences,Laval,Que.)thetwoassayscanbecombinedandruninasinglecapillarytube.PastrapidgenotypingassayshavefocusedononlyonepolymorphismintheVZVgenometodifferentiatewild-typeandvaccinestrainsofVZV(Loparevetal.,2000b).Com-biningthepreviouslydescribedORF62assay(Loparevetal.,2000b)withtheORF38assaydescribedinthisstudy,allowsfortheadditionaldifferentiationoftheEllen(laboratorycontrol)strainofVZV.Runningthesetwoas-sayssimultaneouslyinonecapillaryallowstheinclusionanddifferentiationofthepositivecontrolfromtheclinicalstrains,andthusreducestheriskoffalse-positiveresults.Inconclusion,thisreal-timePCRassayforVZVisarapidandreliablemethodwhichallowsforthesimultaneousdifferentiationofwild-type,vaccineandlaboratorycontrolVZVstrains.Thismethodisusefulforboththeinvestigation 116G.A.Tipplesetal./JournalofVirologicalMethods113(2003)113–116 ofVZV-likeillnessinpreviouslyvaccinatedindividuals,andalsoforlaboratoryconfirmationofVZVinthecontextofsmallpoxbioterrorismlaboratoryinvestigation.Acknowledgements WewishtothankDr.RichardGarceau,Dr.MichaelCar-penter,Dr.MichaelDrebot,Dr.RuntaoHe,Dr.AlbertoSev-erini,BrianKlisko,MichaelGarbuttandAllanGrollaforprovidingvirussamplesforuseinthisstudy.References Argaw,T.,Cohen,J.I.,Klutch,M.,Lekstrom,K.,Yoshikawa,T.,Asano, Y.,Krause,P.R.,2000.NucleotidesequencesthatdistinguishOkavaccinefromparentalOkaandothervaricella-zostervirusisolates.J.Infect.Dis.181,1153–1157. 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