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!-- Point of sampling detection Zika virus within a multiplexed kit capable detecting dengue and chikungunya ( Mixed Material ) --
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mods:abstract Background: Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors.
They cause diseases having similar symptoms in human patients, but requiring different immediate management steps.
Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed.
The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic
acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture,
as this risks contamination of future samples that will give false positives.
Methods: Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito
carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium
groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements
of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed
tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second
mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry
without requiring a continuous chain of refrigeration.
Results: The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika,
chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits
of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and
~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed
to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding
lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human
plasma samples to detect chikungunya and dengue in Pune, India.
Conclusions: A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika,
chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded
fluorescence signals. Assay in dried format allows it to be run in low-resource environments.
Keywords: Point-of-care diagnostics, Multiplexed isothermal amplification, Zika detection, Fluorescence read-out,
Sample preparation, Mosquito surveillance, Virus detection
mods:accessCondition type restrictions on use displayLabel Rights The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
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mods:url access object in context http://ufdc.ufl.edu/AA00054859/00001
mods:name
mods:namePart Ozlem Yaren
Barry W. Alto
Priyanka V. Gangodkar
Shatakshi R. Ranade
Kunal N. Patil
Kevin M. Bradley
Zunyi Yang
Nikhil Phadke
Steven A. Benner
mods:note Yaren et al. BMC Infectious Diseases (2017) 17:293
DOI 10.1186/s12879-017-2382-0; Pages 1-13
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mods:publisher BioMed Central (BMC Infectious Diseases)
mods:dateIssued 2017
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mods:titleInfo
mods:title Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya
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RESEARCHARTICLEOpenAccess PointofsamplingdetectionofZikaviruswithinamultiplexedkitcapableofdetectingdengueandchikungunyaOzlemYaren1,BarryW.Alto2,PriyankaV.Gangodkar3,ShatakshiR.Ranade3,KunalN.Patil3,KevinM.Bradley1,ZunyiYang1,NikhilPhadke3andStevenA.Benner1,4*AbstractBackground:Zika,dengue,andchikungunyaarethreemosquito-borneviruseshavingoverlappingtransmissionvectors.Theycausediseaseshavingsimilarsymptomsinhumanpatients,butrequiringdifferentimmediatemanagementsteps.Therefore,rapid(
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BackgroundZikavirus(genusFlavivirus,familyFlaviviridae)isnativetoAfricaandconsistsofoneAsianandtwoAfri-cangeneticlineages[1,2].Upuntilthelastdecade,Zikaviruspredominantlycirculatedinazoonoticcycleinvolvingforest-dwellingAedesmosquitoesandnon-humanprimatesinAfricaandAsia.Identifiedin1947,Zikainfectionsinhumansremainedsporadicfor~50yearsbeforeemerginginthePacificandtheAmericas[3].AnoutbreakofZikafeveroccurredonYapintheFederatedStatesofMicronesiain2007,andtheninFrenchPolynesiain2013and2014.In2015,ZikavirusemergedforthefirsttimeinBrazil.IthasnowspreadrapidlythroughouttheAmericasalongwiththechikungunyavirus,analphavirusanddenguevirus,anotherflavivirus.TheemergenceofZikaoutsideofAfricahasbeenassociatedwithachangeintransmissionfromapredominantlyzoonoticcycletoatransmissioncycleinvolvinghumanhostsanddomesticmosquitovectors,includingAedesaegyptiandAedesalbopictus.TheseinvasiveAedesspeciessharesimilarecologyandareprimaryvectorsofchikungunyaanddenguevirusesaswell[4,5].TheclinicalpresentationofZikafeverisnonspecificandcanbemisdiagnosed,assymptomsofZikaaresimi-lartoothermosquito-spreadviruseslikechikungunyaanddengue.Amajorityofcasesareasymptomatic(80%,accordingtotheCDC[6]).Inothercases,illnessisclin-icallymildwithsymptomslastingfromseveraldaystoaweek,includingfever,rash,jointpain,conjunctivitis,my-algia,andheadache.SeriousillnessesassociatedwithZikavirusincludeGuillain-Barrsyndromeinadults,microcephalyinneonates,andchronicmusculoskeletaldiseasesthatmaylastmonthstoyears[7].TwocasesarereportedfromNewCaledoniahavingco-infectionofZikaanddengue;Colombiareportsonepatientco-infectedwithZika,chikungunyaanddengue.Thismakesdifferentialdiagnosisevenmorechallenging[8].Sincespecifictreatmentoranapprovedvaccineiscurrentlyunavailable,rapidandreliabledetectionofZikaisneededforinitiationofcontrolandpreventivemeasures,suchasmosquitocontrolandpatientman-agement.Standardserologicalapproaches,suchasantibodydetectionandimmunoassays,oftenhavein-adequatesensitivity.Further,theyarecomplicatedbycross-reactivityinpatientswhohavepreviouslybeeninfectedbyotherflavivirusesfromtheendemicregion[4].Therefore,nucleicacid-targeteddiagnosticsremainasthebestmeanstodetectanddifferentiateZika,chi-kungunyaanddengue.BiologicalconfirmationofZika,chikungunyaandden-gueinfectionsisgenerallybasedondetectionofviralRNAinbloodbyusingreversetranscriptionPCR(RT-PCR)orreal-timeRT-PCRcombinedwithhydrolysisprobes(e.g.TaqManprobes).Inseveralstudies,how-ever,patientswerefoundtogivepositivetestsforZikaintheirsalivaandurine,butnotblood[9,10].Thus,urineandsalivasamplesforZikadetectionarepre-ferredoverbloodbecauseofhigherviraltitersandprolongedpresenceofvirus[11,12].Eventhoughbloodsamplesareshowntohavehigherviralloadsforchikungunyaanddengue,urineandsalivasamplescanstillbeusedtodiagnosethesediseases,especiallyde-sirablefortheireasycollectionandhandling[13,14].RT-PCRdiagnosticsisconsideredthegoldstandardfordiagnostics.However,itrequiresextensivesamplepreparationandexpensiveequipmenttocontrolheat-ingandcoolingcycles.ThismeansthatPCRtestsmustgenerallybeperformedatspecializedfacilities.Apoint-of-samplingnucleicacidtestwouldbevaluableifitreliedonisothermalamplificationratherthanPCR.Thistestcouldbeusedinlowerresourceareas,includingcollegeinfirmaries,doctorÂ’soffices,airportclinics,ambulances,andforward-deployedmilitaryunits.ApowerfulRT-PCRalternative,reversetranscriptionloop-mediatedisothermalamplification(RT-LAMP)usu-allyemploysasetofsixprimersthatbindtoeightdistinctregionswithinthetargetRNA.Itrunsatconstanttemperature,usuallybetween60Cand70(Fig.1a).[15].DuringtheinitialstagesofRT-LAMP,theF2regionofFIPhybridizestoF2cregionofthetargetRNA,andreversetranscriptaseinitiatesthesynthesisofthecomple-mentaryDNAstrand.OuterPrimerF3hybridizestotheF3cregionofthetargetRNAandextends,displacingtheFIPlinkedstrand.Thisdisplacedstrandformsaloopatits5-end.Then,thesinglestrandedDNAwithaloopatthe5endservesasatemplatefortheinternalBIPprimer,whoseB2portionhybridizestoB2cregionofthetemplateDNA.DNAsynthesisistheninitiatedbyastrand-displacingpolymeraseleadingtotheformationofacomplementarystrandandopeningofthe5-endloop.TheouterprimerB3thenhybridizestoB3cregionofthetargetDNAandisextended,displacingtheBIP-linkedstrand.Thisresultsintheformationofadumb-bellshapedDNA.ThedumbbellstructurethenbecomesaseedforexponentialLAMPamplification.Thisamplificationisfurtheracceleratedbytheloopprimers(LFandLB),whicharedesignedtohybridizebetweenF1candF2,B1candB2,respectively[16].Theamplificationproductsincludeconcatemersoftheregionintheanalytethatistargeted,andmayfoldtoaformÂcauliflower-likestructuresÂŽ,whichhavemultipleandrepeatingloops.AlthoughZikadetectionusingRT-LAMParchitecturehasbeenpreviouslyreported,thesemethodologiesarebasedonasingletargetdetection,signalgenerationisYarenetal.BMCInfectiousDiseases (2017) 17:293 Page2of13
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notsequence-specific(e.g.turbiditymeasurementoruseofdsDNAbindingdyes),lacksmultiplexingabilityandcanbedeceivedbyoff-targetamplifications,thereforesusceptibletocreatingfalsepositives[17Â…19].Toenablemultiplexingandreal-timemonitoring,wehavecoupledtarget-specificfluorescentlytaggedstranddisplaceableprobeswithRT-LAMPtodetectZika,chikungunyaanddenguevirusesinbiologicalsamplessuchasurineandplasma,andmosquitocarcassesinfectedwithZikaandchikungunyaviruses.MethodsLaboratorysettingViruspropagationandmosquitoinfectionstudieswereperformedatBSL-3facilityoftheFloridaMedicalEntomologyLaboratoryinVeroBeach,FL.RT-LAMPexperimentswereperformedintheBSL-2laboratorysharedbyFfAMEandFirebirdBiomolecu-larSciencesLLCinAlachua,FL.Patientsamples(wholebloodcollectedinEDTA)suspectedwithchi-kungunyaordenguewerecentrifugedtoseparateplasmainGenePathDxfacility(CausewayHealthcare,Pune,India).AllsamplesthatwereusedforRT-LAMPhavepreviouslybeentestedpositivebyquantitativePCR.PrimersandprobesPrimerdesignwasperformedusingin-housesoftware,Ol-igArchv2(FfAME,Alachua,FL),designedtocreatepri-mersetsthataccountfortheevolutionaryvariationwithinthegenomesofviraltargets.Viralsequencesfordengue-1weredownloadedfromtheBroadInstitute[20],whilethoseforothertargetsweredownloadedfromtheNIAIDVirusPathogenDatabaseandAnalysisResource(ViPR)[21].Multiplesequencealignments(MSAs)werecreatedforthesesequencesusingMUSCLEv3.8.31[22].TheresultingMSAswereusedasinputtoOligArch,whichsearchesforprimersetsthatareconservedwithinatargetofinterestwhileavoidingunintendedtargetsalsoincludedwithintheMSA(allowing,forexample,distinctionbe-tweendenguesubtypes).RulesforLAMPdesignwerefollowedusingcriteriafromtheEikenGenomewebsite[23].DesignedLAMPsetswerecomparedtotheNCBIRNAvirusdatabaseusingNCBIBLAST[24]toeliminatesetsthatwouldcross-react.Setswerefurthercompared,usingin-housesoftwarePrimerComparev1(FfAME,Alachua,FL),to Fig.1aRT-LAMPisinitiatedbyaddinginternalprimers(FIPorBIP)thatannealedbyWatson-Crickcomplementaritytoregions(F2corB2c)withinthetargetRNA.Theouterprimer(F3orB3)thenhybridizestoitsprimingsite(F3corB3c)onthetargetRNAandinitiatestheformationofself-hybridizingloopstructuresbystrandinvasionoftheDNAsequencesalreadyextendedfromtheinternalprimers(FIPandBIP),resultinginadumbbellstructure.RT-LAMPprocesscanbeacceleratedbyloopprimers(LFandLB).bFurther,primingregionofthefluorescentlytaggedprobe(e.g.LB)isextendedbyastrand-displacingpolymerase,andprimerextensionfromthereverseprimersthenreadsthroughtheprimeronthefluorescentlytaggedprobe,displacingtheprobethatbearsaquenchermoiety.Thisseparatesthefluorescentlytaggedoligonucleotidefromthequenchertaggedprobe,allowingthefluorescencetobeobservedinreal-timeandmeasuredfromfluorescentlytaggedprobethathasbeenincorporatedintoRT-LAMPproductsYarenetal.BMCInfectiousDiseases (2017) 17:293 Page3of13
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eliminatesetswithprimersthatwoulddimerizeinamul-tiplexedassaytoproducethefinalsetsofLAMPprimers.LAMPprimersandstranddisplaceableprobeswerepurchasedfromIntegratedDNATechnologies(IDT,Coralville,IA)(Table1).Strand-displaceableprobeswere5-labeledwithFAM,HEX,TAMRAandTETforZika,chikungunya,dengue-1andmitochondrialDNA(positivecontrol),respectively.Quencherprobeswhichpartiallycomplementarytothefluorescentlylabeledprobeswas3-labeledwithIowaBlack-FQ.Alternatively,probestargeting Table1PrimersandstranddisplaceableprobesTargetvirusNameSequence(5-3)LengthStartPosEndPosZikaZV-F3GAGACTGCTTGCCTAG1699059920ZV-B3CTGGGGTCTTGTCTTC1610,14510,130ZV-LFCAGTTGGAACCCAGTCAAC1910,02810,010ZV-LBGTGGAACAGAGTGTGGATTG2010,09310,112ZV-FIPCCATGGATTGACCAGGTAGTTTTTTCGACTGATGGCCAATG41997410,053ZV-BIPACCACTGARGACATGCTTGTTTTTCATGTGGTCGTTYTCC4010,07010,129ZV-LB_NatTailFAMCGGGTTTGCGCTCAGCCATCCGTTCAGTCCGTCAGGTCAG-GTGGAACAGAGTGTGGATTG6010,09310,112ChikungunyaCH-F3CGTCAACGTACTCCTAAC1828912908CH-B3ACGTTGGCTTTRTTTTGG1830943077CH-LFAGCGTCTTTATCCACGGG1829682951CH-LBAYGCATCRATAATGGCGGG1930253043CH-FIPGAAGTTTCCTTTCGGTGGGTTTTTGGAAGACACTYTCYGG4029322993CH-BIPAAGGAGTGGGAGGTGGATTTTTTCAYTTGGTGACTGCAG3930063063CH-LF_NatTailHEXCGGGTTTGCGCTCAGCCATCCGTTCAGTCCGTCAGGTCAG-AGCGTCTTTATCCACGGG5829682951Dengue-1D1-F3ACAGCYCTGAATGAYATGG1995839601D1-B3GCAGTTTCTCTCAGGC1698039788D1-LFCACTTGYTGCCARTCATTCC2096669647D1-LBCCATGCCGYAACCAAG1697279742D1-FIPCTGGTGGAARTGGTGTGATTTTTTGGGAACCTTCAAAAGG4096289693D1-BIPGAAGGAYGGGAGGGAAATAGTTTTTTTAGCCCTRCCCACAAG4297029763D1-LB_NatTailTAMRACGGGTTTGCGCTCAGCCATCCGTTCAGTCCGTCAGGTCAG-CCATGCCGYAACCAAG5697279742MitochondrialDNAMtDNA-F3AGCCTACGTTTTCACAC1791839199MtDNA-B3GCGCCATCATTGGTAT1694109395MtDNA-LBGCCTAGCCATGTGATTTCAC2093229341MtDNA-LFGGCATGTGATTGGTGGGT1892549237MtDNA-FIPGTCATGGGCTGGGTTTTACTTTTTCTACCTGCACGACAAC4092139228MtDNA-BIPCTCAGCCCTCCTAATGACCTTTTTGAGCGTTATGGAGTGG4093599344MtDNA-LB_NatTailTETCGGGTTTGCGCTCAGCCATCCGTTCAGTCCGTCAGGTCAGGCCTAGCCATGTGATTTCAC6093229341Commonquencher CTGACCTGACGGACTGAACGGATGGCTGAGCGCAAACCCG-IowaBlackFQ40AedesaegyptiSSUrRNAAae-F3GGTGTAGTGTGACCTG1625012524Aae-B3GCTAGCTAATGACCAGC1728832866Aae-LBAAGGGCCGGGAAATCG1627772793Aae-LFTCTAAGGGCATCACGGAC1827052687Aae-FIPCGTGCAGCCCAGAACATTTTTGCAAAATGAGATTGAGCG3926602678Aae-BIPCAACGCGTATCCTTGCCTTTTTAATCCCGACTAAATGCG3828202803Aae-LF_NatTail-5IB-FQIowaBlackFQGGGTTTGCGCTCAGCCATCCGTTCAGTCCGTCAGGTCAGTCTAAGGGCATCACGGAC5727052687Aae-LF_NatTail_compFAMCTGACCTGACGGACTGAACGGATGGCTGAGCGCAAACCC-FAM39Underlinedsequencesaredoublestrandsegmentsofstrand-displacingprobes.FAMwasusedforZikadetectionandpositivecontrolforAe.aegyptissurRNAdetection(ex-em=495nm-520nm,colorobservedwithexcitationat470nm,green),HEXwasusedforchikungunyadetection(ex-em=538nm-555nm,colorobservedwithexcitationat470nm,yellow),TAMRAwasusedfordengue-1detection(ex-em=559nm-583nm,colorobservedwithexcitationat470nm,orange),TETwasusedformitochondrialDNAdetectionaspositivecontrolinurine(ex-em=522nm-539nm,colorobservedwithexcitationat470nm,yellow).IowaBlack-FQwasusedasacommonquencherwithabsorptionrangeof420-620nm.Pos:position.SSUrRNA:smallsubunitribosomalRNAYarenetal.BMCInfectiousDiseases (2017) 17:293 Page4of13
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Ae.aegyptismallsubunitribosomalRNA(SSUrRNA)were5-labeledwithIowaBlack-FQand3-labeledwithFAM.Doublestrandportionoftheprobeswerescreenedagainstanyviralgenomesequenceandmosquitogenomicsequence(seeAdditionalfile1:TableS1foradditionalprimersandprobesaspositivecontrols).ViruspropagationandmosquitoinfectionVirusisolatesincludedthefollowing:Zikavirus(PuertoRico),twochikungunyaviruses(LaRunionandBritishVirginIslands)anddengue-1(KeyWest,FL).Allviruseswerepassaged1-3timesinAfricangreenmonkeykidney(Vero)cellsandviraltitersforZikaanddengue-1weredeterminedbyplaqueassay.ChikungunyaviralRNAwasquantitatedusingtheSuperscriptIIIOne-StepqRT-PCRwithPlatinumTaqkitbyInvitrogen(Invitrogen,Carlsbad,CA)asdescribedpreviously[25]withtheCFX96Real-TimePCRDetectionSystem(Bio-RadLaboratories,Hercules,CA)(Table2).InfectionofAe.aegyptifemaleswithZikaandchikun-gunyaviruseswasexplainedindetailinAdditionalfile1.Followinginfection,mosquitolegswereseparatedfromthebodytoconfirmtheinfection.Thelegswereplacedinacentrifugetubewith1mLmedia,twozincbeads,andhomogenizedat25Hzfor3min(TissueLyser:Qiagen,Inc.,Valencia,CA).Thehomogenatewasthenclarifiedbycentri-fugationfor10minat4C.RNAwasthenextractedfromanaliquotofthemosquitoleghomogenate(160L)usingtheQIAampviralRNAminikit(Qiagen,Valencia,CA)andelutedinTEbuffer(50L)accordingtothemanufacturer’sprotocol.ViralRNAwasdetectedusingtheSuper-scriptIIIOne-StepqRT-PCRwithPlatinumTaqkitbyInvitrogen(Invitrogen,Carlsbad,CA)withprimersandprobesspecifictoeachvirus(IntegratedDNATechnologies)(Additionalfile1:TableS2).ProgramsusedforqRT-PCRweredescribedelsewhereforZika[2],chikungunya[26].Legviraltiterswerethendeter-minedbyplaqueassay(Table3).RT-LAMPprocedureReactionmixtures(50Ltotalvolume)containeda10Xprimerset(5L,16MFIPandBIP,2MF3andB3,5MLF(orLBforchikungunya),2MLB(orLFforchikungunya),4MLFquencherprobe,and3MLB-fluorescentprobe(orLFprobeforchikungunya)),deoxynucleosidetriphosphates(dNTPs,1.4mMofeach),Tris-HClbuffer(20mM,pH8.8),KCl(50mM),(NH4)2SO4(10mM,)MgSO4(8mM),Tween20(0.1%),DTT(1mM),Bst2.0WarmStartDNAPolymerase(16U,NEB,Ipswich,MA),WarmStartRTxReverseTranscriptase(15U,NEB,Ipswich,MA),andRNaseOUT™recombinantribonucleaseinhibitor(80U,ThermoFisherScientific,Waltham,MA).TothismixturewasaddedextractedviralRNAs(1L,Zika,chikungunyaordengue-1).Sampleswereincubatedat65Cfor45min,thenanalyzedbyagarosegelelectrophoresis(2.5%)in1XTBEbuffer,followedbyethidiumbromidestaining,usinganappropriateDNAsizemarker(50bpladder;Promega,Madison,WI).FormultiplexedRT-LAMP,each10Xprimerset(5Leach,Zika,chikungunyaanddengue-1)wasaddedinthesamemannertoRT-LAMPmixture(total50Lvolume).RT-LAMPwithurinesamplesInitially,varyingconcentrationsofurine(50%to0%)weretestedinRT-LAMPreactions.Typically,viralRNAspikedurinewasincludedinthereactionmixturetoa10%finalconcentrationwithoutanypurificationstep.Asapositivecontrol,mitochondrialDNAtargetingLAMPprimersweredesignedforuseinurine[27].Similarly,10%salivaandplasmasampleswerealsotestedforZikadetection. Table2VirusesstudiedVirus,Strain(GenBankaccessionnumber)Family/GenusViraltitersZikavirus(ZV),PuertoRico(PRVABC59,KU501215.1)Flaviviridae/FlavivirusGroupIV,positive,ssRNA2.85108pfu/mLChikungunyavirus(CH),BritishVirginIslands(Asianlineage,KJ451624)Togaviridae/AlphavirusGroupIV,positive,ssRNA2.42108genomes/mLChikungunyavirus(CH),LaReunion(IndianOceanlineage,LR2006-OPY1,KT449801)Togaviridae/AlphavirusGroupIV,positive,ssRNA1.89108genomes/mLChikungunyavirus(CH),LaReunionextractedtotalNAfromAedesaegyptifemale(IndianOceanlineage,LR2006-OPY1,KT449801)Togaviridae/AlphavirusGroupIV,positive,ssRNA3.85105genomes/mLDengueserotype1(D-1),KeyWest(FL)(JQ675358)Flaviviridae/FlavivirusGroupIV,positive,ssRNA1.22106pfu/mLpfu:plaqueformingunit Table3ZikaandchikungunyaviraltitersintheinfectedAedesaegyptimosquitolegsZika(ZV)Mosquitoidentity,StrainLegtiterpfu/mLChikungunya(CH)Mosquitoidentity,StrainLegtiterpfu/mLZV3,PuertoRico2.54103CH320,LaReunion1.78104ZV4,PuertoRico9.80102CH328,LaReunion1.51104ZV7,PuertoRico5.10103CH191,LaReunion2.41103ZV9,PuertoRico4.76103CH378,BritishVirginIslands3.21105CH401,BritishVirginIslands4.52104Pfu:plaqueformingunitYarenetal.BMCInfectiousDiseases (2017) 17:293 Page5of13
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Real-timeRT-LAMPForthereal-timemonitoringofRT-LAMP,thereactionswereincubatedat65Cfor60-90minandthefluores-cencesignalsfromFAM-labeledprobeforZika(ex/em=495nm/520nm(usingfilter483-533nm),HEX-labeledprobeforchikungunya(ex/em=538nm/555nm(usingfilter523-568nm),orTAMRA-labeledprobefordengue-1(ex/em=559nm/583nm(usingfilter558-610nm)wererecordedevery30susingRocheLightCycler480(RocheLifeSciences,Indianapolis,IN).Initialreal-timeLAMPexperimentscontainedonly80nMoffluorescentlylabeledLBorLFprobeinsteadof300nM.Finalprimerconcentrationsinthisset-upwereasfollows:1.6MFIPandBIP,0.2MF3andB3,0.4MLFandLB,0.08MLB-fluorescentprobe(orLFforchikungunya)and0.2Mquencherprobe.Finalpri-merconcentrationswith300nMstranddisplacingprobeswereasfollows;1.6MFIPandBIP,0.2MF3andB3,0.5MLF(orLBforchikungunya),0.2MLB(orLFforchikungunya),0.3MLB-fluorescentprobe(orLFforchikungunya)and0.4Mquencherprobe.Additionally,imagesoffluorescencegeneratedbystranddisplaceableprobes,inducedbyblueLEDlight(470nm)atroomtemperature,wererecordedthroughanorangefilterbyacellphonecamera(e.g.iPhone6s).Q-paperbasedRT-LAMPonmosquitosamplesQuaternaryammoniummodifiedpaper(Q-paper)wasmadebytreatingWhatmanfilterpaper(Grade1)withanNaOHsolution,followedbywashingwithwaterandthentreatmentwithglycidyltrimethylammoniumchlor-ide,followingaliteratureprocedure[28].TheQ-papersheetswerecutintosmallsquares(~0.5cm2).Aedesaegyptifemalemosquitoeswerecrushedoneachpapersquarewithamicropestle.ThecrushedcarcassesweretreatedwithaqueousNH3(1M,100L,pH12).Thepaperswerewashedoncewith50%EtOH(100L)andoncewithddH2O(100L),andair-dried.Thepapersquares,withandwithouttargetvirus,werethenplacedinsideRT-LAMPmixtureandincubated65Cfor45min.Priortotestingviruses,aprimerset(aspositivecontrol)targetingAe.aegyptiSSUrRNAwastestedonQ-papercrushednon-infectedmosquitosamples.ManagingforwardcontaminationCarryovercontaminationwaspreventedbyincorpor-ationofdUTPbyBst2.0WarmStartDNAPolymerase(NEB,Ipswich,MA)duringRT-LAMP,andAntarcticthermolabileUDG(NEB,Ipswich,MA)wasusedtodes-troyDNAcontainingdU.Reactionswererunwitha50%inclusionofdUTPmixedwithdTTPgivingfinal0.7mMdTTP,0.7mMdUTP,and1.4mMeachdATP,dCTPanddGTP.AntarcticthermolabileUDG(1L,2units)wasaddedtoRT-LAMPreactionmixture(50L).Sampleswerefirstincubatedat25Cfor5minandthenheatedto65Cfor20-45min.LyophilizationofRT-LAMPreagentsAmixtureofBst2.0WarmStartDNAPolymerase(16U),WarmStartRTxReverseTranscriptase(15U),RNaseOUT™(80U)andAntarcticthermolabileUDG(2U)indialysisbuffer(200L,10mMTris-HClpH7.5,50mMKCl,1mMDTT,0.1mMEDTA10and0.1%TritonX-100)wasplacedinanultrafiltrationmembrane(10kDAcut-offlimit,Millipore,Billerica,MA).Sampleswerecentrifugedat14,000xgforca.15mintoconcentrate(downto~5L)andtoremoveglycerol.10XLAMPprimers(5L),dNTPs(10mMeach,7L)andglycerolfreeenzymemix(5L)werecombinedandlyophilizedandsupplementedwith1.1X-LAMPrehydrationbuffer(22mMTris-HCl,pH8.8,55mMKCl,11mM(NH4)2SO4,8.8mMMgSO4,0.11%Tween20,1.1mMDTT).Plasmasamples(5L)weremixedwith1.1Xrehydrationbuffer(45L)andincubatedat65Cfor45min.TheresultingfluorescencesignalwasobservedbyblueLEDexcitation(470nm)throughanor-angefilter.ResultsModificationsonthearchitectureofRT-LAMPStandardRT-LAMParchitecturefromFig.1awasmodifiedtoimprovethesignaldetectionandtosupportmultiplex-ing.Here,anadditionalcomponentwasaddedintheformofaÂstranddisplaceableprobeÂŽ.ThiscomprisestwoDNAstrandsthatarecomplementaryoverpartoftheirlengths.Thefirstoligonucleotidestrandhasaquenchermoietyatits3-end;thesecondDNAstrandhasafluorophorecova-lentlyattachedatits5-end.Whenthetwostrandsarehy-bridized,thequencherandthefluorophorearebroughtintocloseproximity,andnofluorescenceisobserved.How-ever,the3-portionofthesecondDNAstrandisnotcov-eredbyahybridizingsegmentofthefirstDNAstrand;leftinasinglestrandedform,thisisaprimingsequencecom-plementarytoasegmentoftheloopregionofthedumbbellstructurecreatedbytheinitialstepofRT-LAMP,notonthetargetRNAitself.Further,sincetheprimingsequencehybridizesontheloopregion,thesignaliscreatedonlyaftertheinitialdumbbellisformed.Therefore,itcannotbecreatedbyanynumberofartifactsthatarecommoninRT-LAMP.Thisduplexregionisentirelyunderthecontrolofthedesigner,andneednothaveanyrelationtoanytargetsequence.Further,whenmultiplexingisapplied,samesequencemaybeusedwithdifferentfluorophore:-quencherpairs.DuringRT-LAMP,theprimingregionofthefluores-centlytaggedprobeisextendedbyastrand-displacingpolymerase(Fig.1b).Then,extensionfromthereverseprimersreadsthroughtheprimeronthefluorescentlyYarenetal.BMCInfectiousDiseases (2017) 17:293 Page6of13
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taggedprobe,displacingtheprobethatbearsthequenchermoiety.Thisseparatesthefluorescentlytaggedoligonucleotidefromthequenchertaggedprobe,allow-ingthefluorescencetobeobservedinreal-timeandmeasuredfromfluorescentlytaggedprobethathasbeenincorporatedintoRT-LAMPproducts.TestingRT-LAMPprimersPriortosealedtubeanalysis,theperformanceoftheRT-LAMPprimers(Table1)withthevariousvirustargetsaswellaspositivecontrolsforurineandmosquitosam-pleswasassessedbygelelectrophoresis.ThesamplesweretotalRNAextractedfromviralstocksculturedinAfricangreenmonkeykidney(Vero)cells.Inonecase(forchikungunya),theviralRNAusedwasextractedusingaÂtotalnucleicacidsÂŽpreparationkitfromvirus-infectedmosquitoes(Table2).Thissampleincluded,ofcourse,mosquitoRNAandDNA.Figure2ashowssomerepresentativeresultswithRT-LAMPperformedwiththesesamples.Inbothsingleplexedandmultiplexedcases,theyieldsofLAMPproducts,appearinginagelasaladderofconcatemers,weresimilar.Negativecon-trolsamplesgaveonlythebandsforprimersthemselves,in-cludingthenon-specifictargetcontrolwheretotalnucleicacidextractedfromhealthyAe.aegyptifemalemosquitowasusedasthetemplate.TooptimizeLAMPconditions,magnesiumconcentrationsandoperationtemperatureswerevaried.HighermagnesiumconcentrationsandlowerLAMPtemperaturesgeneratednonspecificamplicons.Therefore,8mMmagnesiumand65Cwereusedinall-subsequentstudies(Fig.2b).Primersetstargetingmito-chondrialDNAinurine,andSSUrRNAinAe.aegyptimosquitoes;aspositivecontrols,yieldedsimilarpatternofLAMPampliconsingelelectrophoresis(Additionalfile2:FigureS1andAdditionalfile3:FigureS2).Gelelectrophoresisrequires,ofcourse,expertpersonnelnotonlytorunthegel,butalsotopreventforwardcontamination,arisingifampliconsfromanearlierassaycontaminatelatersamples,leadingtofalsepositives.Here,thedisplaceableprobegeneratesfluorescencethatcanbereadthroughthewallofasealedtube,whichmayremainsealedthroughouttheassay. AB Fig.2GelelectrophoresisanalysisofRT-LAMPproducts.aTestingRT-LAMPprimersforZikavirus(ZV),chikungunya(CH),anddengueserotype1(D-1)in1-plexand3-plexformats.Misa50bpDNAladder,NTC-z/c/darenotemplatecontrolsforZV,CHandD-1primers.CH-mwastotalnucleicacidpre-extractedfromCH-infectedAe.aegyptifemalemosquito.NSCwasdesignatedasnon-specificcontrolwheretotalnucleicacidextractedfromnon-infectedAe.aegypti.Viraltitersusedwereasfollows:2.85pfuforZV,242genomiccopiesforCH,and1.22pfuforD-1.bTestingdifferentRT-LAMPtemperatures(55Cto70C)andMg2+concentrations(4mMto10mM)inthepresenceofallthreeLAMPprimersforZV,CHandD-1withnotargetRNAYarenetal.BMCInfectiousDiseases (2017) 17:293 Page7of13
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Detectionofvirusesinurinebyreal-timeRT-LAMPFigure3ashowstheresultsfortheLODforZikausingfluorescentprobestargetingZikaRNA.Serialdilutionstudyshowedacleansigmoidalamplificationwith2.85pfuperassay,withasignalriselargelycompleteat20-25min,bothincaseswhereonlyZikaprimerswerepresent(1-plex)orwhenallthreepri-mersetswerepresent(3-plex).Slightlylesssigmoidalcurveswereobservedwith1.425pfu,withsignalgen-erationbeingsubstantiallycompleteafter~30min.Whendilutedfurtherto~0.71pfu,afluorescentsig-nalwasobservedonlyafter~40minforasingle-plexedassay,andafter~50minforthe3-plexedmixture.Inparallel,limitsofdetectionweremeasuredtobe37.8cop-iesand1.22pfuforchikungunyaanddengue-1,respectively(Additionalfile4:FigureS3). ABC Fig.3Real-timeRT-LAMPonZikadetection(ZV).aSingleplexandmultiplexedLODforZikausing80nMFAM-labeledstrand-displacingprobe.Zikaviraltitersrangedfrom2.85pfuto0.71pfu.FluorescentemissionuponirradiationbyablueLED(470nm)wasvisualizedthroughorangefilterglassafterincubationat65Cfor30min.bCross-reactivityassayforZikawherefluorescentprobesforchikungunya(HEX)anddengue-1(TAMRA)wereexcludedandonlyFAM-labeledampliconscouldbedetected.Viraltiterswereasfollows:2.85pfuforZV,242genomiccopiesforCH,and1.22pfuforD-1.Visualizationwasdoneasbeforeafterincubationat65Cfor35min.cTolerancetourineinRT-LAMP.ViraltiterofZikaRNAwas2.85pfu.Varyingconcentrations(0%-50%)ofurineweretestedusing80nMFAM-labeledstrand-displacingprobeYarenetal.BMCInfectiousDiseases (2017) 17:293 Page8of13
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Cross-reactivitywasthentestedusingonlyZika-targetingFAM-labeledprobesinthepresenceofthetwoprimersetsforchikungunyaanddengue-1.Again,astrongsignalwasseenarisingwithin25-30min,butonlyifZikaispresent;chikungunyaordengue-1viralRNAsgavenocross-reactingsignals(Fig.3b).Last,completeZikaviruswasaddedtoanauthentichumanurinesam-pleatincreasingconcentrations(Fig.3c).Presumablybecauseofitselectrolytes,theLAMPsignalwasdelayedfrom15to35minatthehighestratioofurine:buffer(1:1),butnotsubstantiallyatlowerratios.StranddisplacingprobeswerethenintroducedtoRT-LAMPthatallowedthethreevirusestosignalwithdif-ferentfluorescentspecies:fluorescein(FAM)forZika,HEXforchikungunya,andTAMRAfordengue.CompleteviralRNAsextractedfromcellcultureswereusedastargets,andthesewerepresentedin10%urine.Realtimeanalysisoftheemergenceoffluorescenceshowedasubstantialdifferenceforeachtarget(about10mindelays)inthe1-plexcurves(wheretheonlyprimerswerespecificforthetargetvirus)andthe3-plexcurves(whereprimersforalltargetvirusesarepresent).However,inallcases,signalgenerationwaseffectivelycompleteby30min(Fig.4a-c).ThefluorescencewasobservedthroughanorangefilteruponexcitationbyablueLEDemittingit470nm.Thisledtodifferentsignalstrengthsbasedonthedifferentphotophysicsofthethreefluorophores.Thus,theFAMsignalwasthestron-gest,asthe470nmexcitationlightisclosesttothemax-imumoftheFAMexcitationspectrum(Fig.4d).TovisualizeallthreecolorswithoutchangingtheLED,theamountsofprobeswereincreasedfrom80nMto300nM.TheresultsareshowninFig.5a-c.However,increaseintheprobeconcentrationresultedinca.20mindelaytoobtainfluorescentsig-nalinthe3-plexedformat.Inallcases,FAM-labeledprobesforZikawerevisualizedasbrightgreen,HEX-labeledprobesforchikungunyawerevisualizedasgreen-yellow,andTAMRAlabeledprobesfordengue-1werevisualizedasorangewhenexcitedwithblueLED(470nm)andfilteredthroughorangeglass(Fig.5d).Q-paperbasedRT-LAMPoninfectedmosquitosamplesAsapartofmosquitosurveillance,asquareofQ-papercarryingmosquitocarcassesinfectedwithZikaor ABCD Fig.4aSingleplexandmultiplexdetectionofZika(ZV)viralRNA(2.85pfu)in10%urineusing80nMstrand-displacingprobeusingRocheLightcycler(channel483-533),bSingleplexandmultiplexdetectionofchikungunya(CH)viralRNA(242copies)in10%urineusing80nMstrand-displacingprobeusingRocheLightcycler(channel523-568),cSingleplexandmultiplexdetectionofdengue-1(D1)viralRNA(1.22pfu)in10%urineusing80nMstrand-displacingprobeusingRocheLightcycler(channel558-610),dFluorescentemissionuponirradiationbyablueLED(470nm)wasvisualizedthroughor-angefilterglass.Eachviruswasassignedtoadifferentfluorophoretag;FAMforZika,HEXforchikungunya,andTAMRAfordengueYarenetal.BMCInfectiousDiseases (2017) 17:293 Page9of13
PAGE 10
chikungunya(Table3),followingwashingwithammoniaandethanol,couldbedirectlyintroducedintotheLAMPmixturewithoutnegativeeffect(Fig.6).Again,visualfluorescencesignalwasgeneratedwithin30min(Additionalfile5:FigureS4).DiscussionTherecentZikaoutbreakshowstheimportanceoftimelydiagnosisofviraldiseasesatplaceswherepatientspresentwithsymptoms(pointsofsampling).Italsoshowsthechallengesfacedbypublichealthservicestaffastheysurveytheenvironmentformosquitoesthatmightbecarryingarboviruses.Inneithercasedothepractitionerswanttosendasampleawayandwaitforresults.Thekitdevelopedhereprovidestheneededcapabil-itiesinbothsettings.RT-LAMP,runat65C,provedtobeespeciallyconvenient,asZikaandotherviruseslosesallinfectivityattemperatureshigherthan60C[29].Further,RT-LAMPisshowntotoleratemanylowmo-lecularweightsubstancesinbiologicalsamples[30].ThisallowsviralRNAstobeLAMP-amplifiedwithoutapre-viousRNAextractionorpurificationstep.ForZika,thelevelofvirusintheurineofapatienthavingacurrentinfectioniswellabovethelimitsofde-tection(LODs)possibleinthisassay.Further,thelevelofvirusinaninfectedmosquitocapableoftransmittingthevirusarealsowellabovetheLODÂ’sreportedhere.Therelevantlevelsinurineofdengueandchikungunyaarealsodetectableintheseassays[31,32].Therefore,thesensitivityofthiskitisappropriateforallthreepathogens.Tomaketheassayeasytouse,pre-preparedtubescontaininglyophilizedreagentsweredistributedwithanobservationboxthatusesa470nmemittingLEDandanorangefilter(Additionalfile6:FigureS5).Thisisop-timalforthefluorescein(FAM)fluorophore,usedheretotagZikaamplicons.ItislessoptimalfortheHEXandTAMRAfluorophoresthatwereusedforchikungunyaanddengue,respectively.Thus,thelasttwovirusesarelesseasilydetectedbyhumaneyethanZika,eventhoughtheamplificationprocessappearstobenodifferent. ABDC Fig.5aSingleplexandmultiplexdetectionofZika(ZV)viralRNA(2.85pfu)in10%urineusing300nMstrand-displacingprobeusingRocheLightcycler(channel483-533),bSingleplexandmultiplexdetectionofchikungunya(CH)viralRNA(242copies)in10%urineusing300nMstrand-displacingprobeusingRocheLightcycler(channel523-568),cSingleplexandmultiplexdetectionofdengue-1(D1)viralRNA(1.22pfu)in10%urineusing300nMstrand-displacingprobeusingRocheLightcycler(channel558-610),dFluorescentemissionuponirradiationbyablueLED(470nm)wasvisualizedthroughor-angefilterglass.mtDNA(humanmitochondrialDNA)servedasapositivecontrol(TET-labeledstrand-displacingprobe).Eachviruswasidentifiedwithadif-ferentflorescenttag,FAMforZika,HEXforchikungunya,andTAMRAfordengueYarenetal.BMCInfectiousDiseases (2017) 17:293 Page10of13
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Itisknownthatsomeintercalatingdyes(e.g.SYBRGreenI)mayinhibittheLAMPorPCRreactions[33].Delayinthesignalgenerationmightbeattributedtohigherprobeconcentration,especiallyinthe3-plexedreactions.Inonecase,however,whentheamountofBst2.0DNApolymerasewasdoubled,improvementonthetimetosignalwasobserved(datanotshown).Ontheotherhand,increaseintheprobeconcentrationledtovisualizationoffluorescentsignalgeneratedbythepres-enceofeachtargetvirus.Alternatively,delaysinthesignalgenerationmightbemitigatedbyadditionoftwonewLEDsthathaveemis-sionsmoreappropriatetoexcitethetwootherfluoro-phores.ThiswillbenecessaryifhighermultiplexingisdesiredtopickupadditionalarbovirusessuchasoÂ’nyongnyongandMayaro.Suchhighermultiplexingmayalsorequiretheuseofstrategicallyplacedalterna-tivenucleicacidanalogs,suchastheself-avoidingmo-lecularrecognitionsystemsdescribedinthe22-plexforarbovirusesreportedbyGlushakovaetal.[34].InadditiontohavinganarchitecturethatneverrequirestheassaytubetobeopenedafterthetargetRNAisamplified,forwardcontaminationwasmitigatedbyasecondexpedient.ThisreplaceddTTPbyamixtureofdTTPanddUTP,leavingto2-deoxyuridinebeingincorporatedintotheamplicons.Thismakestheampli-consthetargetsfordestructionbyauracil-DNAglyco-sylase(UDG).Thus,thermolabileUDGdigestsanysurvivingampliconsatroomtemperatureastheLAMPsamplesarebeingsetup,preventingyesterdayÂ’sampliconsfrombeingtodayÂ’scontaminants.Further,todeploythiskitinlowresourcelocations,anyglycerolpresentincommerciallyacquiredenzymeswasremovedbyultrafiltration,asolutioncontainingdNTPsandLAMPprimerswasadded,andthemixturewasfreeze-driedintubesthatwererehydratedonlocationtoruntheassay.Accordingtotheliterature,thesepathogenscanalsobedetectedinsalivaandplasma.Totestthiskitwiththesebiofluids,samplesofplasmaandsalivawerespikedwithZikaviralRNAandaddedtothemixtureina1:9ratioofsample:buffer(Additionalfile7:FigureS6).ThisworkwasrepeatedinIndiausingplasmasamplesfrompatientsinfectedwithchikungunyaanddengueexploit-ingthelyophilizedreagentkitshippedwithoutrefriger-ation.Fluorescentsignalwassuccessfullygeneratedwithin30min(Additionalfile8:FigureS7).Anotherneedforimmediatedetectioninvolvesmos-quitosurveillance.Forexample,inHaiti,whenahouse-holdisfoundtocontainanindividualinfectedwiththevirus,mosquitoesinandaroundthathouseholdarerou-tinelycollected.Theseusuallyhavelowerpriorityforpublichealthresources,soaninexpensivemultiplexedkittosurveythemwouldhavespecialvalue.Thus,weaskedwhetherthiskitwouldworkonmosquitocar-cassesthathadbeencrushedonpapercontaininghighlevelsofcovalentlyimmobilizedquaternaryammoniumsalts(Q-paper).Q-paperhastheadvantageofcapturingallthenucleicacidsevenifthemosquitocarcassistreatedwithasterilant(suchasaqueousammonia)orwashed(for Fig.6WorkflowforZikadetectiononinfectedmosquitosamplesusingQ-papertechnology.Amosquitobody(ZV9,Table3)wasfirstcrushedonQ-paperandtreatedwith1MaqueousNH3(pH12)solution,andpaperwasthensequentiallywashedwith50%EtOHandwater.Q-papercontainingmosquitosamplewerethendippedintoRT-LAMPmixtureandincubatedat65Cfor30minandfluorescentsignalgeneratedwasvisualizedusingLEDbluelight(470nm)throughorangefilterglass.TheimagewasrecordedbycellphonecameraYarenetal.BMCInfectiousDiseases (2017) 17:293 Page11of13
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example,withethanol,toremovefluorescentcompoundsfromthemosquitocarcass,suchaspterins[35,36],whichmightinterferewithin-tubeanalysis).WewereconcernedthattheQ-paperwoulditselfinhibitRT-LAMP.However,wefoundthatappropriatelysizedQ-papercarryingmos-quitocarcassescouldbedirectlyintroducedintoRT-LAMPmixture,andtheamplificationwasstillsuccessful(Additionalfile9:FigureS8).ConclusionsAkit,completewithavisualizationapparatusandrefrigeration-freesampletransport,isnowavailableforrapidpoint-of-samplingdetectionofZika,chikungunya,anddengueviruses.Bydirectlyaddingviruscontaminatedurine,plasmasamplesorsquaresofQ-papercontaininginfectedmosquitoestoRT-LAMPmixtures,aminimumdetectionlevelsof~0.71pfuequivalentviralRNAsforZika,~1.22pfuequivalentviralRNAsfordengue,and~38copiesofchikungunyaviralRNA,wereachieved.Theassayisreadin20-40minbyvisualizing(humaneye)three-colorcodedfluorescencesignals.Whenpre-mixedreagentsandenzymesarelyophilizedinthetubestobeusedintheassays,thetubescanbedistributedtolowerresourcesettingswithoutrefrigeration.AdditionalfilesAdditionalfile1:Document_PointofSamplingDetectionofZikaViruswithinaMultiplexedKitCapableofDetectingDengueandChikungunya(DOCX111kb)Additionalfile2:FigureS1.GelelectrophoresisofLAMPprimerstestedforhumanmitochondrialDNAinurine.Notemplatecontrols(NTCs)wereperformedintheabsenceofurinesample,1-plexor3-plexNTCsshowednoladderlikeamplicons.Inthepresenceof10%urine,allprimersetsbothin1-plexand3-plexformats,gaveladderlikeamplicons(JPEG33kb)Additionalfile3:FigureS2.GelelectrophoresisofRT-LAMPprimerstestedonsmallsubunitrRNAoffemaleAe.aegyptimosquitoes.CrushedspecimenswereeitherputdirectlyintoRT-LAMPmixture,orfirstcrushedonQ-paperandthenwentthroughammoniatreatmentpriortoRT-LAMP.Ineithercase,set2failedtogotocompletionwhereasforset1,mostoftheprimerswereconsumedwithin30minofincubationat65C.Notemplatecontrolexperimentsdidnotproduceanyampliconasexpected(JPEG44kb)Additionalfile4:FigureS3.Limitofdetectionfor1-plexchikungunyaanddengue-1RT-LAMPexperiments.SubstratesforthisexperimentwereextractedviralRNAfromVerocellcultures.(A)VaryingtitersofchikungunyaviralRNAs(~189to18copies)wereincludedinRT-LAMPreagentsandrunreal-timeusingLightcycler(channel523-568).Forchikungunyadetection,80nMofHEX-labeledprobeswereused,andabout38copiesofchikungunyaviralRNAcouldbedetectedinlessthan30min.(B)Varyingtitersofdengue-1viralRNAs(~2.44to0.12pfuequivalentRNAcopies)wereincludedinRT-LAMPreagentsandrunreal-timeusingLightcycler(channel558-610).Fordengue-1detection,80nMofTAMRA-labeledprobeswereused,andabout1.22pfuequivalentcopiesofdengue-1aviralRNAcouldbedetectedwithin35min(JPEG58kb)Additionalfile5:FigureS4.Gel-electrophoresisandvisualizationofRT-LAMPproductswithLEDbluelight(excitationat470nm)throughorangefilter.(A)DetectionofZika(ID#3and4)andchikungunya(ID#320and328)in3-plexformatwithinfectedmosquitolegsorbodies.Zikainfectedmosquitosgeneratedbrightgreenfluorescence(FAM-labeledprobe)whereaschikungunyainfectedmosquitoesgeneratedyellow-greenfluorescence(HEX-labeledprobe).GelelectrophoresisanalysisshowedthatinthepresenceoftargetviralRNA,ladderlikeampliconsweregenerated.(B)VisualizationofZika-infected(ID#7and9)andchikungunya-infected(ID#191)mosquitosamplesin3-plexformatonQ-paperafterRT-LAMPrunat65Cfor30min.ZikasamplesgeneratedbrightgreensignalduetoFAM-labeledprobeswhereaschikungunyacontainingsamplesgeneratedmorelikeyellow-greensignalduetotheuseofHEX-labeledprobes(JPEG56kb)Additionalfile6:FigureS5.ThisobservationboxisnowavailableforpointofsamplingrapiddetectionofZika,chikungunya,anddengue.Thisboxusesa470nmemittingLEDbluelightandanorangefilterwithasingleAAbatteryalreadyembedded(JPEG40kb)Additionalfile7:FigureS6.GelelectrophoresisanalysisofZikadetectioninsalivaandblood.LikeurineRT-LAMPexperiments,extractedZikaviralRNAs(2.85pfu)werespikedwithsalivaandplasmasamples,and10%finalconcentrationofsalivaorplasmawasincludedintoRT-LAMPmixtures.Zikapositivesampleswereidentifiedasladder-likeampliconsonagarosegel(JPEG21kb)Additionalfile8:FigureS7.(Top)Real-timeRT-LAMPofchikungunyasamplesusingRotorGeneQ(Qiagen,Germantown,MD,USA).UsingdryformatRT-LAMP,9plasmasamplesand1purifiedRNAsampleweretestedinreal-timeandfluorescentsignalsweregeneratedwithin30minforallcases.(Bottom)RT-LAMPondenguesamples(plasma)wastestedandsignalgenerationwasobservedbydetectionboxfromAdditionalfile6:FigureS5(JPEG46kb)Additionalfile9:FigureS8.Gel-electrophoresisofRT-LAMPprimerstestedonZikaorchikungunyainfectedfemaleAe.aegyptimosquitoes(Table3)crushedonQ-paperandwentthroughammoniatreatment.Zika-infectedAe.aegypti(ID#7)andchikungunya-infectedAe.aegypti(ID#378)samplesonQ-paperwererunin1-plexformatwhereaschikungunyainfectedmosquito(ID#401)wasrunin3-plexformatwhereallprimersforZika,chikungunyaanddengue-1werepresentintheRT-LAMPmixture.Allsampleswithpresentedviruswereabletogenerateladderlikeampliconswithin30minofincubationat65C(JPEG38kb)AbbreviationsLAMP:Loop-mediatedisothermalamplification;LOD:Limitofdetection;MSAs:Multiplesequencealignments;Q-paper:QuaternaryammoniumfunctionalizedWhatmanpaper;RT-LAMP:Reversetranscriptionloop-mediatedisothermalamplification;SSUrRNA:smallsubunitribosomalRNA;UDG:UracilDNAglycosylaseAcknowledgementsDengue-1virus(strainBOL-KW010)waskindlyprovidedbytheFloridaDepartmentofHealthBureauofLaboratories.ZikavirusandtheAsianlineageofchikungunyavirusweregraciouslyprovidedbytheCentersforDiseaseControlandPrevention.TheIndianOceanlineageofchikungunyaviruswaskindlyprovidedbyRobertTesh(WorldReferenceCenterforEmergingVirusesandArboviruses,throughtheUniversityofTexasMedicalBranchinGalveston,Texas)totheUF-FMEL.WethankS.Bellamy,B.Eastmond,S.Ortiz,D.Velez,K.Wiggins,R.Zimler,andK.Zirbelforassistancewiththeinfectionstudies.WearealsoindebtedtotheDTRAbasicresearchprogram,theNIAID,andtheStateofFloridaforongoingsupport.FundingTheworkwassupportedinpartbyHDTRA1-13-1-0004andNIAID1R21AI128188-01.TheprojectwassponsoredinpartbytheDepartmentoftheDefense,DefenseThreatReductionAgency.Thecontentoftheinformationdoesnotnecessarilyreflectthepositionorthepolicyofthefederalgovernment,andnoofficialendorsementshouldbeinferred.ResearchreportedinthispublicationwassupportedinpartbytheNationalInstitutesofHealth.ThecontentissolelytheresponsibilityoftheauthorsanddoesnotnecessarilyrepresenttheofficialviewsoftheNIH.AvailabilityofdataandmaterialsThedatasupportingtheconclusionsofthisarticlewillbeavailablefromthecorrespondingauthoruponrequest.AuthorsÂ’contributionsOYdesignedandrantheexperiments,andcollectedandanalyzedthedate.OYandSABdraftedthemanuscript.BWApreparedthesamplesofmosquitoesthatYarenetal.BMCInfectiousDiseases (2017) 17:293 Page12of13
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wereinfectedbytheviruses,andprovidedextractedviralRNAs.KMBdesignedtheprimersandprobes.PVG,SRR,KNPandNPrantheexperimentsonplasmasamplesinIndia.OY,BWAandSABallrevisedthemanuscript.ZYprovidedadviceconcerningthedirectuseofQ-paper.Allauthorseditedandapprovedthefinalmanuscript.CompetinginterestsSeveraloftheauthorsandtheirinstitutionsownintellectualpropertyassociatedwiththisassay.ConsentforpublicationNotapplicable.EthicsapprovalandconsenttoparticipateMosquitoesweretheonlyanimalsdirectlyusedinthisstudy.Theprocedurestomanagechickens,whosebloodwasusedtofeedtheinfectedmosquitoes,wereapprovedasIACUCProtocol#201507682bytheUniversityofFloridaInstitutionalAnimalCareandUseCommittee.Publisher’sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.Authordetails1FoundationforAppliedMolecularEvolution(FfAME),Gainesville,FL,USA.2FloridaMedicalEntomologyLaboratory,UniversityofFlorida,VeroBeach,FL,USA.3GenePathDx(CausewayHealthcare),Pune,Maharashtra,India.4FirebirdBiomolecularSciencesLLC,Alachua,FL,USA.Received:30December2016Accepted:4April2017 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• We accept pre-submission inquiries  Our selector tool helps you to nd the most relevant journal We provide round the clock customer support  Convenient online submission Thorough peer review Inclusion in PubMed and all major indexing services  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Yarenetal.BMCInfectiousDiseases (2017) 17:293 Page13of13
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