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Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya

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Title:
Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya
Creator:
Ozlem Yaren
Barry W. Alto
Priyanka V. Gangodkar
Shatakshi R. Ranade
Kunal N. Patil
Kevin M. Bradley
Zunyi Yang
Nikhil Phadke
Steven A. Benner
Publisher:
BioMed Central (BMC Infectious Diseases)
Publication Date:
Language:
English

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Dengue ( jstor )
Viral RNA ( jstor )
Viruses ( jstor )

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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
General Note:
Yaren et al. BMC Infectious Diseases (2017) 17:293 DOI 10.1186/s12879-017-2382-0; Pages 1-13

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University of Florida
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University of Florida
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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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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
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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
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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
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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,doctor’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,andmayfoldtoaformcauliflower-likestructuresŽ,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,anadditionalcomponentwasaddedintheformofastranddisplaceableprobeŽ.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),theviralRNAusedwasextractedusingatotalnucleicacidsŽpreparationkitfromvirus-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

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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