Two CSFV genome copies were detected using the standard protocol in each used PCR-machine; 20 genome copies were the detection limit with the high-speed approach in the cyclers from Bio-[r]
(1)ContentslistsavailableatSciVerseScienceDirect
Journal ofVirological Methods
j o ur na l h o me p a g e :w w w e l s e v i e r c o m / l o c a t e / j v i r o m e t
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Rapiddetectionof foot-and-mouthdisease virus,influenzaAvirus andclassical swinefever virusbyhigh-speedreal-time RT-PCR夽
KerstinWernike,MartinBeer,BerndHoffmann∗
InstituteofDiagnosticVirology,Friedrich-Loeffler-Institut(FLI),Suedufer10,17493Greifswald-InselRiems,Germany
Articlehistory:
Received29January2013
Receivedinrevisedform7May2013 Accepted13May2013
Available online 20 May 2013 Keywords:
Foot-and-mouthdisease InfluenzaA
Classicalswinefever Real-timePCR High-speedPCR
a b s t r a c t
Highsensitivity,minorriskofcross-contaminationandinparticulartherapidreactiontimemake quan-titativereal-timepolymerasechainreaction(qPCR)assayswellsuitedforoutbreakinvestigationsaswell asformonitoringepidemicsofpathogens
InthisstudyqPCRassaysforthreehighlycontagiousanimaldiseases,namelyfoot-and-mouth-disease (FMD),influenzaA(IA)andclassicalswinefever(CSF)havebeendeveloped.Furthermore,an amplifi-cationcontroltargeting18SribosomalRNAwasincluded.Eachassaywasvalidatedwithsamplesfrom infectedanimalsusingthreedifferentstandardqPCR-machinesintwothermalprofiles:onestandardand onehigh-speedapproach,respectively.Thehigh-speedPCRassaysallowedthereliablediagnosisofFMD, influenzaAandCSFinlessthan28minwithananalyticalsensitivityofatleast200genomecopies/lin everycase,withslightdifferencesregardingreactiontimeandsensitivityfortheindividualPCR-cycler instruments.Therefore,thenewlyestablishedrapidRT-PCRsystemswillbeavaluablemethodforthe monitoringandcontrolofthesethreeimportantvirusesandwillbearobustoptionforthedevelopment ofnovelmolecularpen-sidetests
© 2013 The Authors Published by Elsevier B.V All rights reserved
Foot-and-mouthdisease(FMD),influenzaAaswellas classi-calswinefever(CSF)arehighlycontagiousdiseasescausinghigh economiclossesinanimalproductionworldwide.Topreventthe spreadofthesediseasesintohugegeographicareas,rapid labo-ratorydiagnosisiscrucial(Belak,2007).Duetothecombination ofahighsensitivity,thereducedriskofcross-contaminationand thepossibilityofquantitativeanalysis,real-timePCR(qPCR)isa valuabletoolforthedetectionofviruses(Mackay,2004;Mackay etal.,2002).However,usedcommonlyPCR-protocolsrequireabout 90–120min.Todecreasethetimetakenfordiagnosishigh-speed qPCRassaysfordetectionofe.g.influenzaoradenoviruseshave beendevelopedrecently(Fujimotoetal.,2010;Sakuraietal.,2011) ThedescribedPCRsystemsprovidedaresultinlessthan30min,but aspecializedqPCR-machinewasused.Inthisstudy,high-speedPCR assaysforthedetectionoffoot-and-mouthdiseasevirus(FMDV), influenzaA viruses(IAV)and classical swinefevervirus(CSFV) havebeendevelopedandvalidatedusingthreedifferentcommon standardqPCR-machines
夽 Thisis anopen-accessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense,whichpermitsunrestricteduse,distributionand reproductioninanymedium,providedtheoriginalauthorandsourcearecredited
∗Correspondingauthor.Tel.:+493835171201;fax:+493835171193 E-mailaddress:bernd.hoffmann@fli.bund.de(B.Hoffmann)
Avarietyofdifferentsamplematerialsobtainedfromanimals infected experimentallywithFMDV,IAV andCSFVrespectively, wasused in this study.Allsamples wereprovided by the cor-responding German National Reference Laboratories RNA was extractedwiththeQIAamp®ViralRNAMiniKit(Qiagen,Hilden,
Germany)accordingtothemanufacturer’srecommendationsand elutedin50lkitelutionbuffer
Primer and probes specific for CSFV and IAV have been
describedpreviously(Hoffmannetal.,2005,2010).Toselect FMDV-specificprimersandprobespublishedsequenceinformation(NCBI database)wasused.In additiontothepathogen-specificassays eachsamplewastestedbyaninternalamplificationcontrol(IC) specificPCRsystemwhichtargets18SribosomalRNA.Sequences ofallprimersandprobesareshowninTable1
TheRT-qPCRswerecarriedoutusingtheSuperScriptTMIII One-Step RT-PCR Systemwith Platinum® Taqin a total reaction of
12.5l,theamountofmastermixturecomponentsfortheFMDV-, IAV-,CSFV-andIC-specificPCRsystemsisgiveninTable2 Concen-trationsofprimersandprobesnecessaryinasinglereactionare listedinTable1.Themergingofthesinglemastermixture com-ponentswasexecutedatroomtemperature.Finally,2.5lRNA template wasaddedand qPCRwascarried outusing aBio-Rad CFX96Real-TimeDetectionSystem(softwareversionBio-RadCFX Manager2.0;Bio-Rad,Hercules,CA,USA),anEcoTMReal-TimePCR system(EcoTM Softwarev3.0.16.0;Illumina,Inc., SanDiego,CA, USA)aswellasaLightCyclerđ480Real-TimePCRSystem(LCS480 0166-0934/$seefrontmatterâ 2013 The Authors Published by Elsevier B.V All rights reserved
(2)Table1
Sequencesofprimersandprobesusedinthisstudy
Name Sequence5–3 Concentration(pmol/reaction) Amplicon(basepair) Reference
FMD-IRES-3F ACCTGGWGRCAGGCTAAGGA 10 78 Thisstudy
FMD-IRES-3.2R CCYRGTCCCCTTCTCAGAT 10 FMD-IRES-3FAM FAM-CCCTTCAGGTACCCCGAGGTAACA-BHQ1 3.125
CSF100-F ATGCCCAYAGTAGGACTAGCA 10 93 Hoffmannetal.(2005)
CSF192-R CTACTGACGACTGTCCTGTAC 10
CSF-Probe1 FAM-TGGCGAGCTCCCTGGGTGGTCTAAGT-BHQ1 1.25
IAV-M1-F AGATGAGTCTTCTAACCGAGGTCG 10 99 Hoffmannetal.(2010)
IAV-M1.1-R TGCAAAAACATCTTCAAGTYTCTG 7.5
IAV-M1.2-R TGCAAAGACACTTTCCAGTCTCTG 7.5
IAV-M1-FAM FAM-TCAGGCCCCCTCAAAGCCGA-BHQ1 1.25
18sRNA-1F GCGGGTAACCCGTTGAACC 156 Thisstudy
18sRNA-1R CCATCCAATCGGTAGTAGCG
18sRNA-1FAM FAM-ATTCCCCATGAACGAGGAATTCCCAGTA-BHQ1
1.5.0.39;Roche DiagnosticsDeutschland,Mannheim, Germany) Thefollowingthermalprofilewasused:PCRinitialactivationstep at95◦Cfor 1min;45 cyclesof atwo-stepcycling consistingof denaturationat98◦Cfor1sandannealingandextensionat54◦C for1s.Aseparatereversetranscriptionstepwasnotperformed.The shorttimebetweenPCRmixpreparationandTaqpolymerase acti-vationbytheinitialdenaturationstepwassufficientforaneffective cDNAgeneration.Inordertocomparetheresultswithastandard method,an identicalreverse transcription qPCR (RT-qPCR) set-upwascarriedoutinthethreeqPCR-machinesmentionedabove usingthefollowingthermalprofile:reversetranscriptionstepat 50◦Cfor15min,PCRinitialactivationstepat95◦Cfor2min;45 cyclesofathreestepcyclingconsistingofdenaturationat95◦C for15s,annealingat56◦Cfor20s,andextensionat72◦Cfor30s Allsamplesweretestedinduplicateswithanappropriateexternal standard
Thetwodifferentthermalprofilesresultedinatotalreaction timeof1hand42min(standardprotocol),and40min,23s (high-speedPCR)whenusingtheBio-RadCFX96Real-TimeDetection System.WiththeEcoTMReal-TimePCRsystemthetimerequired togetafinaldiagnosiswas1h,29min,and27minand55s, respec-tively;andwiththeLightCycler®480Real-TimePCRSystem1hand
37minor33minand44swerenecessary(Fig.1)
TheanalyticalsensitivityoftheFMDV-,IAV-andCSFV-specific PCRsystemswasdeterminedusingseriesof10-folddilutionsofan appropriatestandard(Fig.2).TheFMDassayamplifiedtheRNAina linearfashionfrom2.0E+06copiesdownto2copies/lusingboth thermalprofilesineverycycler(Figs.1and2).Differentsample typesobtainedfromcattle,goats,sheeporswineinfected exper-imentallywithFMDVstrainsofserotypesA,C,OandAsiawere testedbothwiththestandardandthehigh-speedprofile.The FMD-aswellastheIC-specificPCRsystemgaveapositiveresultineach case(Table3)
WhenusingthestandardprotocoltheIAVassayamplifiedRNA downto2genomecopies/l,andidenticalresultwasobtainedwith
Table2
Compositionofmastermixturesforeachofthetargets
Reagent Volume(l)
FMDV CSFV IAV IC
2×reactionmix 6.25 6.25 6.25 6.25
5mMmagnesiumsulfate – 1
RT/Platinum®TaqMix 0.25 0.25 0.25 0.25
RNase-freewater 1.5 2.5 1.5 –
Primer-probe-mix 1 1
NegativeRNAswine – – – 1.5
TemplateRNA 2.5 2.5 2.5 2.5
Totalreactionvolume 12.5 12.5 12.5 12.5
thehigh-speedPCRintheBio-RadCFX96Real-TimeDetection Sys-tem.UsingtheEcoTMReal-TimePCRsystemortheLightCycler®
480Real-TimePCRSystemincombination withthehigh-speed approach,20genomecopies/lcouldbedetected(Figs.1and2) Withexceptionofonethroatswab(subtypeH1N1)testedinthe LightCycler®480everydiagnosticsampleshowedapositiveresult
forIAVandICintherapidthermalprofile(seeTable3)
TwoCSFV genomecopies were detectedusing thestandard protocolineachusedPCR-machine;20genomecopieswerethe detectionlimitwiththehigh-speedapproachinthecyclersfrom Bio-RadandIllumina,and200genomecopies werethelimitin theinstrumentfromRoche(Figs.1and2).Serumsamples,tonsils andlymphnodesofswineandwildboarinfectedwithdifferent genotypesweretestedlikewise.Everysamplewasfoundpositive whenusingtheBio-RadCFX96Real-TimeDetectionSystemand theEcoTMReal-TimePCRsystem.However,thehigh-speed proto-colincombinationwiththeLightCycler®480failedtodetecta2.3
strainintonsilsofawildboar,inthestandardprotocolthissample scoredpositivewithaquantificationcyclevalueof30.2(1.2E+03 genomecopies/l).Inaddition,5outof14sampleswerepositive inonlyoneofthetestedduplicates.IneverycasetheIC-specific assaygaveapositiveresult(Table3)
Insummary,allviruspositivediagnosticsamplesrepresentinga broadrangeofviralgenomeloadsweredetectedbythehigh-speed PCRbothontheCFX96systemfromBio-RadandtheEcoTM Real-TimePCRsystemfromIllumina(100%diagnosticsensitivity).With
(3)Table3
Diagnosticsamplesusedinthisstudy.InvestigationofeverysamplewascarriedoutinthreedifferentPCR-machinesusingtwothermalprofiles,oneofthemastandard protocol,theotherahigh-speedapproach
Samplematerial Serotype/subtype/strain (genotype)
Bio-RadCFX96 EcoTMReal-TimePCRsystem LightCycler®480
Standardprotocol 1h,42min
High-speed PCR40min 23s
Standard protocol1h, 29min
High-speed PCR27min 55s
Standard protocol1h, 37min
High-speedPCR 33min,44s
Cq Cq IC Cq Cq IC Cq Cq IC
Foot-and-mouthdiseasevirus
Serumcattle A(A22) 23 25 22 22 25 22 22 25 28
Serumgoat Asia 29 31 22 29 32 22 28 32 28
Salivacattle O(O1/Manisa) 19 22 22 20 22 23 17 23 28
Salivacattle Asia 18 21 21 18 22 22 16 22 27
Nasalswabcattle O(O1/Manisa) 20 23 22 21 23 21 18 24 28
Nasalswabgoat A(A22) 34 37 22 33 37 22 32 39 28
Lingualvesiclecattle C(C1/Oberbayern) 20 26 17 21 27 19 19 28 25
Intranasalvesiclecattle C(C1/Oberbayern) 17 23 21 17 24 21 16 25 25
Footvesicleswine C(C1/Oberbayern) 14 21 21 15 21 21 12 23 24
Footvesiclesheep O(O1/Manisa) 19 23 19 19 22 22 17 24 24
Footvesicleswine A(IRN/97) 17 22 21 18 22 23 15 23 25
Serumcattle C(C1/Oberbayern) 23 27 22 23 27 22 21 29 29
Nasalswabcattle C(C1/Oberbayern) 24 28 22 25 29 22 22 30 27
Salivacattle C(C1/Oberbayern) 21 25 22 22 26 22 19 27 27
Lingualvesiclecattle C(C1/Oberbayern) 23 27 21 24 28 22 21 30 27
InfluenzaAviruses
Throatswabs H5N1 25 29 22 27 32 22 28 37 29
Throatswabs H5N1 22 25 23 23 28 22 24 34 30
Throatswabs H5N1 20 24 22 22 27 22 23 33 28
Throatswabs H5N1 17 21 22 19 23 22 20 29 29
Throatswabs H5N1 21 25 22 23 28 22 24 34 28
Cloacalswabs H5N1 26 31 22 28 32 22 29 39 28
Cloacalswabs H5N1 29 33 22 31 36 22 32 43 28
Cloacalswabs H5N1 23 27 20 24 29 22 25 35 24
Cloacalswabs H5N1 29 33 22 30 35 22 31 42 28
Cloacalswabs H5N1 28 33 21 29 35 22 31 42 30
Throatswabs H1N1 31 33 22 32 36 23 33 42 27
Throatswabs H1N1 35 37 22 36 39 24 38 noCq 29
Throatswabs H1N1 35 36 22 35 39 25 38 45a 28
Throatswabs H1N1 31 34 21 31 36 23 33 42 27
Throatswabs H1N1 28 31 21 28 33 24 30 38 32
Throatswabs H1N1 30 33 23 30 35 24 33 39 29
Classicalswinefevervirus
Serumswine Losten/Freese98(2.3) 27 32 23 27 32 27 29 33a 28
Serumswine Losten/Freese98(2.3) 25 30 22 24 32 24 26 36a 28
Serumswine Losten/Freese98(2.3) 33 38 22 33 38 30 35 39a 29
Serumswine Losten/Freese98(2.3) 28 33 22 29 33 24 29 36 29
Serumswine Losten/Freese98(2.3) 27 31 22 27 32 23 29 36 29
Serumswine Losten/Freese98(2.3) 32 37 24 33 37 22 34 38a 25
Serumswine Losten/Freese98(2.3) 23 28 23 22 28 25 25 34 40
Serumswine Koslov(1.2) 13 19 22 13 19 22 15 22 28
Serumswine Koslov(1.2) 20 27 22 20 27 22 22 30 27
Tonsilswildboar Rösrath(2.3) 29 43 21 28 36 31 30 noCq 32
Lymphnodewildboar Rösrath(2.3) 27 34 17 26 33 20 28 38 23
Tonsilswildboar Rösrath(2.3) 29 37 18 28 35 21 31 41 23
Lymphnodewildboar Rösrath(2.3) 24 33 15 23 32 19 26 35 20
Tonsilsswine Rösrath(2.3) 22 30 17 21 29 20 24 32 24
Lymphnodewildboar Rösrath(2.3) 21 29 17 20 27 21 22 33a 22
Cq,quantificationcycle;IC,internalcontrol aPositiveresultinonlyoneoftheduplicates.
theLightCycler®480fromRoche44of46clinicalsamplesscored
positive(95.8%diagnosticsensitivity),butinsomecasesonlyone oftheduplicateswaspositive
Forhighlycontagiousanimaldiseasesrapiddiagnosisis essen-tialtopreventthespreadintofurtheranimalpopulations.Suitable laboratorymethods areso-calledrapid-cycleor high-speedPCR assays; results are provided in less than 1h (Wittwer et al., 2001).ThosePCRsystemshavebeendescribedrecentlyforvarious pathogens suchas Vibriocholerae, groupBStreptococcus bacte-ria,influenzavirusoradenoviruses(Fujimotoetal.,2010;Koskela etal.,2009;Mölsäetal.,2012;Sakuraietal.,2011;Wilsonetal., 2010).However,inmostcasesspecializedqPCR-machineswere
used.Animportantargumentfortheapplicationofthoseportable cyclerswasthe possibleusagein the field orin regional labo-ratories(Mưlsäetal.,2012).However,theEcoTMReal-TimePCR systemandBio-RadCFX96Real-TimeDetectionSystemwithits weightof13.6kgand21.4kg,respectively,aretransportable eas-ilytoregionallaboratoriesoraffectedanimalfarmsaswell.The LightCycler® 480(55kg)maybeusedpreferentiallyin
(4)Fig.2.AnalyticalsensitivityoftheFMDV-,IAV-andCSFV-specificPCRsystems basedon10-folddilutionseriesofpositivestandardRNA.Ineachcase,thestandard protocolisdepictedbyanunfilledbarandtheresultsofthehigh-speedPCRare symbolizedbyafilledbar
Thedifferenttimeperiodsnecessaryfortheidenticalthermal profilesinthethreethermalcyclerswerebasedmainlyonthe per-formanceofthePeltierelementsusedandthetimeforfluorescence datacollection.However,ineverycasearesultcouldbeexpected inlessthan45min.TheverygoodresultsoftheBio-RadCFX96 Real-TimeDetectionSystemareobviouslytheconsequenceofthe longerthermalcycling.Itislikelythatanincreaseofcycling condi-tionsfortheLightCycler®480similartotheCFX96systemwould
improvedthesensitivityalsoforthissystem.Ontheotherhand theexcellenthigh-speedPCRresultsoftheEcoTMReal-TimePCR systemusingtheshortestthermalprofileinthetestconfirmthe possibilityforaneffectivespeedingupofPCRprotocolsingeneral NewtechnologieslikethePeltierelementoftheEcoTMReal-Time PCRsystemforaveryfastrampingcombinedwithintuitive soft-warepackageswillopenupthepen-sidediagnosticmarketalsofor real-timePCRtechnology
Asshowninthisstudy,thesensitivityoftheindividual detec-tionsystemsvarieddependentonthecombinationofPCRassays andcyclerinstruments.TheFMD-specificassayinthehigh-speed approachamplifieddownto2genomecopiesusingboththeEcoTM Real-TimePCRsystemandtheBio-RadCFX96Real-Time Detec-tion withnearlyidentical quantificationcycle values compared to thestandard protocol On the other hand,the CSFV-specific assaytestedwiththeLightCycler®480displayeda100-foldlower
analyticalsensitivityinthehigh-speedcomparedtothestandard thermalprofile.Inaddition,thealternativeassaysspecificforCSFV orIAVgavenegativeresultsforeverydiagnosticsample,andonly one outof five tested FMDV-specificsystems achievedan ade-quate sensitivity inthehigh-speed approach (datanot shown) Hence,well-establishedqPCRassaysandthechemistryusedfor reaction requireappropriate validation regarding suitabilityfor high-speed approaches Thereafter, genome detection for every pathogen should be possible in less than 45min using qPCR-machinesalreadyavailableinthelaboratory
Inconclusion,thenewlydevelopedhigh-speedPCRsystemsare asensitiveandrapidmethodforconfirmingthepresenceofFMDV, IAVorCSFVgenomesandwillbeavaluablemethodfortherapid monitoringofwithinoutbreakdiagnosisormonitoring/screening approachesinspecializedaswellasregionallaboratories.An appli-cationinthefieldwithoutthenecessityforshippingthesamples toaspecializedlaboratoryseemsalsotobefeasible
Acknowledgements
This work was funded by Zentrales Innovationsprogramm Mittelstand(ZIM)oftheFederalMinistryofEconomicsand Tech-nology.EcoTMReal-TimePCRsystemandLightCycler® 480were
kindlyprovidedbyLTFLabortechnik(Wasserburg,Germany)and Roche Diagnostics Deutschland (Mannheim, Germany), respec-tively
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