Quantitative Dengue Serotyping: The Development of a ... · Serotyping: The Development of a Higher Performance Method Using SYBR Green Assay. Arch Clin Microbiol. Vol. 8 No. 4:55

2017Vol. 8 No. 4:55

Research Article

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ARCHIVES OF CLINICAL MICROBIOLOGYISSN 1989-8436

iMedPub Journalswww.imedpub.com

DOI: 10.4172/1989-8436.100055

Salles TS1,2, Sá-Guimarães TE1, Souza DFS1, López SBG1, Alvarenga ESL1, Franco TA1, Melo ACA1,3, Soares MR1, Ferreira DF2,4 and Moreira MF1,3*

1 DepartmentofBiochemistry,InstituteofChemistry, Federal University of Rio de Janeiro, 21941-909, Rio de Janeiro, RJ, Brazil

2 DepartmentofVirology,InstituteofMicrobiology, Federal University of Rio de Janeiro, 21941-909, Rio de Janeiro, RJ, Brazil

3 NationalInstituteofScienceandTechnologyinMolecularEntomology,Brazil

4 DepartmentofMolecularandStructuralBiochemistry,NorthCarolinaStateUniversity,NorthCarolina,USA

*Corresponding author: Moreira MF

[email protected]

Department of Biochemistry, Federal UniversityofRiodeJaneiro,Av.AthosdaSilveiraRamos149Room536-A,RiodeJaneiro, RJ, 21941-909, Brazil.

Citation: SallesTS,Sá-GuimaraesTE,SouzaDFS,LópezSBG,AlvarengaESL,etal.(2017)QuantitativeDengueSerotyping:TheDevelopmentofaHigherPerformanceMethodUsingSYBRGreenAssay.ArchClinMicrobiol.Vol.8No.4:55

Quantitative Dengue Serotyping: The Development of a Higher Performance

Method Using SYBR Green Assay

AbstractDenguefeverisaseriousdiseasethatthreatens40%oftheworld’spopulation,andwithoutavaccineortherapy,accuratediagnosisiscriticalintryingtosavepatients.Ourworkestablishesanalternativeandmorespecificsetofoligonucleotidesforuseindetectingthedengueviralgenomeviasemi-quantitativenestedPCRusinga relatively inexpensive enzyme and a quantitative PCR (qPCR) method usingSYBRGreen. To validate theDENVdetectionmethods, assayswereperformedtoensurethespecificityandsensitivitybygeneratingfiveampliconsofstandardvirussamplesobtainedfrominfectedVerocellcultures.Semi-quantitativenestedPCR and SYBRGreenPCR techniques are important tools in the detection andquantificationofthefourserotypesofdenguevirusandcanbeusedfordenguevirusdetectioninmosquitovectors,aswellinserologicalorotherhumanfluids,inmuchthesamewayastestsfordose-responsivenessofantiviralsinlaboratorycellculturesandviralloaddeterminationsareperformedinothertypesofstudies.Keywords: Denguevirus;DENVDetection;PCR;SYBRGreen;Serotyping

Received: July28,2017; Accepted: August05,2017;Published: August12,2017

IntroductionDengue feverarthropod-bornedisease is transmittedbyAedes andcausedbydenguevirus(DENV),amemberoftheFlavivirus genus and Flaviviridae family. Dengue fever is currently oneof themost importantepidemicssince it threatens40%of theworld'spopulationwholiveinhighriskareas.Everyyear,50to100millioncasesofDenguefeverarereportedworldwide,with12,500deaths[1-5].

Atpresent,Brazilfaces3simultaneousepidemicscausedbytheZikavirus(ZIKV),Chikungunyavirus(CHIKV)andDENV,inadditionto some cases of sylvan yellow fever virus (YFV) infections, allofwhicharetransmittedbytheAedes aegyptimosquito.Inthiscontext, the clinical and epidemiological diagnoses of thesediseasesaredifficultsincetheirsymptomsareverymuchalike[6].

AdefinitivediagnosisofDENVinfectiondependsonisolatingthevirusanddetectingantigensorviralRNA[11].Thereare4dengueserotypes (DENV1-4), which are genetically similar and shareapproximately65%oftheirgenomes.Thisdiagnostictesttakestime,makingmedicaldecisionsdifficult.Anaccurateandurgentdenguediagnosisisalsoessentialforadequateepidemiologicalcontrol by the Disease Control Measures Program [7,8]. The

mostefficientapproachtoconfirmthepresenceofarbovirusesisthroughmoleculardiagnostics.TheWorldHealthOrganizationrecommendstheuseofgenomicdetectionmethodsforDENVsforlaboratoryconfirmationofthevirusduringthefivetosixdaysaftertheonsetofsymptoms[9,10].PCR-basedtechniques,suchas polymerase chain reaction (PCR) and reverse transcriptionPCR (RT-PCR) are suitable tools for the identification and

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determination of different dengue serotypes during the acuteperiod of the disease [11,12]. In general, accurate PCR resultsusing this protocol are incompatible with inexpensive enzymekits, which led to a search for cheaper ways to detect DENV1-4sinceBrazil facesa scarcityofeconomic resourcesand thedengueoutbreakcannotbeignored.

Chien et al. [13] reported assessing the performances andadvantagesof4 standard semi-quantitativePCRassays for thedetectionofdenguevirusRNAinclinicalsamples.Theyupgradeda C-prMprotocol,firstdescribedbyLanciotti,etal.[14]throughthemodificationoftheDENVconsensusD1(mD1)andserotype-specificTS2(mTS2)primersandredesignedtheserotype-specificTS1 (rTS1)andTS4 (rTS4)primers.Theauthors concluded thatthe newly established C-prM protocol was the most sensitivemethodtodetectandquantifydengueserotypes[15].

OurworkestablishesanewmethodandalternativeapproachforserotypingdengueviruswithmorespecificsetofoligonucleotidesdesignedneartheregiondescribedbytheChienetal.[16].Wepropose an experimental procedure using semi-quantitativenested PCR using a less expensive enzyme and a quantitativePCR (qPCR) method using SYBR Green. To validate the DENVdetectionmethod,assayswereperformedtoensurespecificityand sensitivitywith regard to five amplicons of standard virussamplesobtainedfrominfectedVerocellcultures.

Material and MethodsOligonucleotide designArepresentativesequenceinNCBIfromeachviruswasusedforthedevelopmentof theprimers; specifically,DENV-1 (Hawaii),DENV-2 (16681), DENV-3 (H87), DENV- 4 (H241), Japaneseencephalitisvirus (Sa14-14-2),St. Louisencephalitisvirus (MSI-7), West Nilevirus (NY99), YFV (17D) and ZIKV (MR766) wereused.ThesesequenceswerealignedusingtheClustalWtoolonthe website (http://www.genome.jp/tools/clustalw/) to designadequate DENV primers. This step was followed by a secondalignment using only DENV sequence regions similar to thosedescribedbyLanciottietal. [17] todesigngeneralandspecificprimers. Based on the DENV1-4 alignment, it was possible todetermine the best primer sequences following the guidelinesdescribed by Thornton and Basu, (2011). In order to check ifdesignedprimerpositionsareconservedinDENVvirussequencesinC-prMregion,wefirstsearchedthenucleotideNCBIdatabaseusing keyword expression: "Dengue virus"[porgn: _txid12637]ANDgenomeNOTpartialNOTnearl”.Itwasfound4,089completegenomesequencesofDENV1-4.Thesesequenceswerejoinedinafilenameddengue_genomes.fasta.Asasecondstep,weused theembossprimersearchtool locallytosearchtheDNAdatabase(dengue_genomes.fasta)formatchwiththesetsofspecificprimersfor DENV1-4, separately. Primers (DENV1=STD1/rTS1; DENV2=STD2/mTS2;DENV3=STD3/TS3andDENV4=STD4/rTS4)

Preparation of virusesInthiswork,thefourDENVserotypes,aswellasZIKVandYFVwereused.Allviruses(MOI=0.02)weregrowninAfricangreenmonkeykidney(Vero)epithelialcellsthatwereinitiallygrownin25cm²plasticculturebottles,followedbypassagingina750cm²

rollerbottlesystemandwereincubatedat37°Cfor7daysuntilobservationofcytopathiceffects.ThesupernatantsofeachDENVserotype were removed, centrifuged, aliquoted and stored at-80°C.Subsequently,virusesweretitratedbytheplaquemethodas described and by Putnaketal.(1998)withmodificationsandwereculturedat37°Cwith5%CO2inDulbecco'smodifiedEagle'smedium (DMEM; InvitrogenCarlsbad,CA,USA), supplementedwith 2.5% fetal bovine serum (Gibco-Invitrogen, Carlsbad, CA,USA),100units/mLpenicillinand100μg/mLstreptomycin [18].TheseDENV(1-4)sampleswereusedforthepositivestandards.

Viral RNA extraction and cDNA synthesisTotalRNAwasextractedfrom200µLofviralsupernatantsusingthe TRIzol method (Invitrogen, Carlsbad, CA, USA) accordingto the manufacturer's instructions, followed by elution in 25μL of nuclease-free water. The viral RNA concentrations wereestimatedat260nmusingaSmartSpecspectrophotometer(Bio-Rad,Hercules,CA,USA).For theDNase treatment,8μLof theviralRNAsample,1μLofDNaseI10xbufferand1μM RNase-free DNase(Fermentas,Burling,Canada)wereincubatedfor30minat37°Cfollowingthemanufacturer’sinstructions.Thereactionswere stopped with 50 nmol of ethylenediaminetetraaceticacid at 65°C for 10min. The cDNAwas then synthesized fromthe DNase-treatedmRNA using a High-Capacity cDNA ReverseTranscription Kit (AppliedBiosystems, Foster City, CA, USA)accordingtothemanufacturer'sprotocol.Afterwards,thecDNAconcentrationswereestimatedat260nm[19]usingaSmartSpecspectrophotometer(Bio-Rad,Hercules,CA,USA).

Semi-quantitative nested PCRDENVserotypingbysemi-quantitativenestedPCR involvestwoamplification reactions of the target sequence, similar to thatdescribedbyLanciottietal.[7]Inthefirststep,a10μLreactionmix containing 1 μL of cDNA, 100 nmol of the STD and D2primersandPhusionhigh-fidelityDNA polymeraseKitreagents(ThermoFisher,Waltham,MA,USA)wereaddedaccordingtothemanufacturer's protocol. Amplification involved the followingsteps:aninitialdenaturationandactivationofthePhusionDNApolymeraseat98°Cfor3min,followedby35cyclesof98°Cfor5s,55°Cfor5s,72°Cfor20s,andafinalextensionfor10minat72°C.Inthesecondstep,thehighefficiencyPhusionhigh-fidelityDNApolymerase (ThermoFisher,Waltham,MA,USA)wasusedwithHFbuffercontaining1μLoftheabovereactionproductand50nmolofonesetofprimers(STD1/rTS1;STD2/mTS2;STD3/TS3andSTD4/rTS4)ina20μLreactionvolume.Amplificationinvolvedthefollowingsteps:aninitialdenaturationandactivationofthePhusionDNApolymeraseat98°Cfor3min;followedby25cyclesof98°Cfor10s,55°Cfor10s,72°Cfor30s;andafinalextensionfor10minat72°C.Following theamplification,a10μLaliquotof each amplicon product was analyzed by 1.5% agarose gelelectrophoresisandtheDENVserotypesweredeterminedbythespecificsizeampliconaspresentedinTable 1.

Synthesis and extraction of plasmidsAmplicons generated with the STD and D2 primers for eachDENV serotype, all of which were 572 bp, were cloned usingthepGEM-TEasyIvectorsystem(Promega,Fitchburg,WI,USA)

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includedintheqPCRutilizedthefollowingsteps:adenaturationat94°Cfor1mfollowedby78.5°Cfor10sandarampto94°Catarateof0.1°C/10swithcontinuousfluorescencemeasurement.

Data analysis (absolute quantification)After amplification by the qPCR assay, data acquisition froma CFX96 Touch Real-Time PCR Detection System (Bio-RadLaboratories,Hercules,Calif.,USA)wasperformed.A standardcurve using cDNA obtained from infected cells (DENV1-4) andotherswithclonedplasmidDNAsequences(copynumber)werecorrelatedtothresholdcycle(CT).TheCTisdefinedasthecycleat which a statistically significant increase in Rn (normalizedreportersignal)isfirstdetected.ThetargetcDNAorcopynumberoftheclonedplasmidandtheCTvalueareinverselyrelated[21].Thecalibrationcurvesofthecopynumberobtainedfromplasmidsamples were correlated with the CT values, and then thesevalueswereusedwiththecDNAsamplesofeachcorrespondingvirustoquantifythegeneticmaterial,thusrelatingthenumberofcopiesofDENV1-4presentinthecDNAtotheCTvalue.

For the statistical analyses, the CT values were exported to aMicrosoftExcelworksheet.RegressionanalysesoftheCTvaluesofthecDNAsamplesorclonedplasmidserialdilutionswereusedtodeterminetheamplificationefficienciesandsensitivityofthemethod[22].

ResultsStandard nested PCR normalizationTo standardize the PCR, the primers were first designedaccordingtoinitialcriteria.Then,theC-prMgeneregionofeachDENVserotypewasselectedbecauseitshowspointsofgenomicconsensusinsidealargeregionoflowsimilarity[23].Analignmentof some FlaviviruseswasperformedtocheckthedesignoftheprimerstoensurethatonlyDENV1-4andnootherFlaviviruses wouldbeamplified(datanotshown).FortheconservedregionoftheDENVs,onepairofprimerswasdesignedtoidentifyaregion

Name of Primers and gene region SequencePRIMERSa Specific viral Set of primers Size (pb)C-prM STD c TTTATTTAGAGAGCAGATCTCTG DENV-all (F)

STD-D2 572D2b TTGCACCAACAGTCAATGTCTTCAGGTTC DENV-all (R)

STD1c ACGGGTCGACCGTCTTTCAA DENV-1 (F)STD1-rTS1 225

rTS1b CCCGTAACACTTTGATCGCT DENV-1 (R)STD2c GCGAAAAACACGCCTTTCAA DENV-2 (F)

STD2-mTS2 140mTS2b CGCCACAAGGGCCATGAACAGTTT DENV-2(R)STD3c ACGGGAAACCGTCTATCAA DENV-3 (F)

STD3-TS3 302TS3b TAACATCATCATGAGACAGAGC DENV-3 (R)

STD4c GTGGTTAGACCACCTTTCAA DENV-4 (F)STD4-rTS4 282

rTS4b TTCTCCCGTTCAGGATGTTC DENV-4 (R)C-prM STDc TTTATTTAGAGAGCAGATCTCTG DENV-all (F)

STD-rmD1 89rmD1c CGGTTTCTCTCGCGTTTCAGCATATTGA DENV-all (R)

a.ThepositionofthegenomeisbasedonDenguevirustype3.b.PrimersdevelopedbyLanciottietal.1992andChienetal.2006.c.Primersdevelopedinthisstudy.

Table 1 CharacteristicofDenguevirusprimersintheC-prMgenefortheSYBRGreenassayprotocol.

followingthemanufacturer’sinstructions.Theplasmidcontainingeach amplicon product was transformed into the bacteriumEscherichia coli (E. coli) strainDH5-αbyheat shock, incubatingthecellswithplasmidsfor30minat4°C,followedbya90sheatshockat42°C,thena10minrestat4°C.Then,thetransformedE. coliwasplacedin800μLofLuria-Bertani(LB)broth(LBBrothUltrapure,AffymetrixInc.,Cleveland,OH,USA)madeaccordingto the manufacturer's protocol and incubated in a shaker for1hour at 37°Cat 100 rpm.After this incubation, thebacterialsuspensionwasplatedontosolidLBagar(Ultrapure,AffymetrixInc., Cleveland,OH,USA) containing 100 μg/mL ampicillin andmade according to the manufacturer's protocol. The resultingcolonies were screened by colony PCR using the T7 and SP6primers. Positive colonieswere grown in liquid lysogenybrothcontaining 100 μg/mLampicillinat37°Covernight,asdescribedbyFigueira-Mansuretal.[20].

Quantitative PCR (qPCR)For the qPCR amplification, the viral cDNA of each serotypeand the primer sets (STD1/rTS1; STD2/mTS2; STD3/TS3 andSTD4/rTS4)wereseparatelyusedfortheidentificationofDENVserotypes.Ineachprotocol,thePowerSYBRGreenMix(AppliedBiosystems,FosterCity,CA,USA)wasusedfollowingthereactionconditionsandpost-amplificationanalysisrecommendedbythemanufacturer. Primers for the four serotypes (DENV1=STD1/rTS1;DENV2=STD2/mTS2;DENV3=STD3/TS3andDENV4=STD4/rTS4) were used separately in each well of a qPCR plate. Thereactionshadfinalvolumesof15μL,andcontained5μLofcDNAin several dilutions, 2.5 μL of each primer set (400 nmol) and7.5μLofreactionmixture.Amplificationinvolvedthefollowingsteps:50°Cfor2min;95°Cfor10min;and40cyclesof94°Cfor30s,50°Cfor30s,72°Cfor60s.SincetheSYBRGreenmaynon-specifically intercalate into double-stranded DNA during PCR,a melting temperature (Tm) curve analysis was performed toconfirmtheidentityoftheamplifiedproductthroughitsspecificTmprofile(Morrisonetal.,1999).TheanalysesoftheTmcurves

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AimingtocheckifthedesignedprimerpositionsareconservedinDENV1-4sequences,weusedprimersearchtoolasdescribedinMaterialandMethods.Intheoreticalmatchanalysis,allDENVserotypesprimersetswerespecificuntilanerrorrateofatleast20%. DENV 1 and 4 with sequences of dengue_genome fastadatabase(4,089completesequences),matchedonlywithDENV1and4, respectively, and their geneticvariantswithanerrorof

thatwascommontoallDENVstobeusedforasemi-quantitativenestedPCR. Fourpairsof primersweredesigned insideof thecommon region of each DENV1-4 sequence for the detectionof viral genomic material. In Figure 1, the alignmentof partialsequencesofDENVserotypesisshown,withforwardandreverseprimershighlighted, the lastofwhichwerealsousedby Chien etal.[24]toidentifycommonandspecificregionsofDENV1-4.

Figure 1 MultiplenucleotidesequencealignmentoftheDENVs: Thealignedregioncorrespondstonucleotides56to654ofDENV.The50%majorityconsensussequenceislistedasthereferencetohighlighttheconservationof this region. In addition to the four viruses used in this study, the virusesDENV-1(Hawaii),DENV-2(16681),DENV-3(H87),DENV-4(H241)arealigned.TheDENVconsensusprimers(STDandD2)andfourvirus-specificforwardprimersarehighlighted inboldfaceanditalicized;thefourvirus-specificreverseprimersfromChienetal.(2006)arehighlightedinboldfaceandunderlined.

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20% (data not shown). This result suggests that these sets ofprimersarespecific todetectofDENV1and4. In theanalysisforthesetofprimerstodetectDENV2and3,theresultswereevenbetter,onlyDENV2and3, respectively, and their geneticvariants werematched considering an error of 30% (data notshown). The matching analysis above showed that the set ofprimersdesigned,inthiswork,wasaccuratetodetectDENV1-4.TheTable1showsthesetsofprimerswithallthecharacteristicsofeachDENVserotype.

Upondeterminationoftheviraltiter,aliquotscontaining200µLofeachDENVserotypeweresubjectedtoviralRNAextraction,from which cDNA was subsequently synthesized and usedfor conventional nested PCR using the primers in Table 1, as described in Material and Methods. Figure 2 shows that eachspecific pair of primers for the DENV serotypes amplified onesingle PCRproduct corresponding to each viral serotype cDNAusingthissemi-quantitativemethod.

Quantification and analytical sensitivity of SYBR green using plasmid DNA as templateFor sensitivityanalysesandPCRquantificationusingSYBRGreen,theseriallydilutedplasmidsofDENV1-4wereusedastemplateandamplifiedusingeachspecificprimerset(STD1/rTS1;STD2/mTS2;STD3/TS3andSTD4/rTS4).Dilutionseriesoftheplasmidstandards contained 4.54 × 106 copies of DENV-1, 7.29 × 106 copiesofDENV-2, 3.38×106 copiesofDENV-3and3.62×106 copiesofDENV-4.The linear relationshipbetween the startingplasmidDNAandtheCTvaluesformedtheregressioncoefficients(R2), which were greater than 0.99 for almost all viruses. The

meanvaluesof triplicateCTsrangedfrom:11.03±0.045(4.54×106copies)to30.73±0.045(4.54copies)ofDENV-1;14.87±0.078(7.29×106copies)32.79±0.078(7.29copies)ofDENV-2;11.58±0.134(7.29×106copies)32.75±0.135(7.29copies)ofDENV-3;and14.87±0.098(7.29×106copies)35.74±0.098(7.29copies)ofDENV-4,asshowninFigure 3A and Table 2A.Thus,thecoefficientofvariationwaslessthan4.0%forDENV1-4,similartowhatwasdescribedbyDharetal.[22].

Standardization of SYBR green-based qPCR using cDNATo determinetheefficiencyoftheqPCRassay,cDNAsfromthe viral serotypes and the specific primers described in Table 1 were used. First, the cDNA was tested using theprimers in concentrations ranging from 100-500 nmol foreach primer set (STD1/rTS1; STD2/mTS2; STD3/TS3 and STD4/rTS4),withviralserotypecDNAsdiluted1:10.Theprimerswereusedata concentrationof400nM,and the cDNAswereusedin 10-fold serial dilutions, as described in Table 2B. Then, the analytical sensitivity of the SYBR Green PCR was determinedthroughassessingthe linearrelationshipbetweenthedilutionsof theviral cDNAand theCTvaluespresented inFigure 3B. In thisanalysis,weobtainedtheregressioncoefficients(R2),whichweregreaterthan0.99foralmostallviruses.Theequationsofthelineswerecalculated,andtheslopesobtainedwereusedtodetermine theefficiencyof the SYBRGreen reactionusing theformula: ,whereα is the angular coefficient of theline [25].Inthisway,thevaluesofamplificationefficiencieswiththeprimerssetsintheSYBRGreenassaysweredetermined,withallvaluesbeing higherthan90%forallDENVserotypes,asshownin Table 3.

Figure 2 PCRoftheDENV1-4positivetemplates: Agarosegelaftersemi-quantitativenestedPCRamplificationwiththespecificprimersetsusingtheDENV1-4cDNAastemplate,wheretheacronymsstandfor:MM=molecularmassstandard,B=negativecontrolreaction,DV1=cDNAofdenguevirus1,DV2=cDNAofdenguevirus2,DV3=cDNAofdenguevirus3,DV4=cDNAofdenguevirus4.

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Specificity of amplification against control virusesTo confirm the specificity of each set of primers in the SYBRGreenPCRassay,viralcDNAofDENV1-4fromanewlypassagedof cultures cell was used with their specific and non-specificsets of primers for detection. Samples of viral ZIKV and YFV

cDNA, both also belonging to the Flaviviridae family, wereincludedintheassay.Table4showsthemeansoftriplicateCTvalues corresponding to assays using viral cDNA (1:100) withspecific and non-specific primer sets. When amplification wasundertaken for DENV1, the DENV1 cDNA sample significantlyincreased inSYBRGreenfluorescence,withameanCTvalueof28.18,whereas SYBRGreenfluorescencewasnotdetected in

Figure 3 qPCR calibration curve:ThestandardcurvesforDENV-1(A),DENV-2(B),DENV-3(C)andDENV-4(D)obtained

bySYBRGreenPCRusingplasmidDNAastemplate.ThenumberofcopiesofDENVplasmidDNAaddedtoeachreactionmixturewasderivedfrominaserial10-folddilutionforeachtemplate,startingfromtheconcentrationintermsofthenumberofcopies(4.54×106copiesforDENV-1,7.29×106copiesforDENV-2,3.38×106 copies forDENV-3and3.62×106 copies forDENV-4) corresponding to thenumbers in logbase10on the linearcurveinpanelsA-D.ThestandardcurvesforDENV-1(E),DENV-2(F),DENV-3(G)andDENV-4(H)obtainedbySYBRGreenPCRusingviralcDNAastemplate.Thesecurvesweremadefromserial10-folddilutionsofeachtemplate(correspondingtothenumbersinlogbase10onthelinearcurveinpanelE-H)(4.54× 106 copies for DENV-1,7.29×106copiesforDENV-2,3.38×106copiesforDENV-3and3.62×106copiesforDENV-4).

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the other viral cDNA samples. Likewise, theuseof cDNA fromDENV2-4 significantly increased in SYBR Green fluorescence,withmeanCTvaluesof32.79,32.90and31.28,respectively,forspecificviralcDNAs,withnoSYBRGreenfluorescencedetectedwhenothercDNAwastested.

Since the SYBR Green PCR involves no post-PCR analysis, theamplificationofspecificvs.non-specificproductswasconfirmedbyananalysisofthedissociationcurvesofthetargetamplicons.TheamplificationprofilesandthedissociationcurvesforDENV-1,DENV-2,DENV-3,DENV-4,togetherwithnegativeviralcontrolsofYFVandZIKV,areshowninFigure 4.Inaspecificreactiontodetect DENV-1, cDNA from the DENV-2, DENV-3 and DENV-4

serotypeswasusedasanegativecontrol,andasimilarstrategywas used for all serotypes, as determined for each specificreaction.

IntheSYBRGreenqPCRanalysistodetectDENV-1,theDENV-1cDNAexhibitedasignificantincreaseinthefluorescenceofSYBRGreen,withameanCTvalueof28.18(Table 4 and Figure 4A). In addition,theamplificationofDENV-2,DENV-3,DENV-4,YFVandZIKVcDNAdidnotyieldanysignificantincreaseinfluorescence,indicating an absence of the specific DENV-1 target. Whenamplification was performed to detect DENV-2, the DENV-2cDNAexhibitedasignificantincreaseinthefluorescenceofSYBRGreen,withameanCTvalueof32.79(Table 4 and Figure 4C). The amplificationofDENV-1,DENV-3,DENV-4, YFV and ZIKV cDNAdidnotsignificantlyincreasefluorescence,indicatinganabsenceof the DENV-2 target. When amplification was performed todetectDENV-3,theDENV-3cDNArecordedasignificantincreaseinthefluorescenceofSYBRGreen,withameanCTvalueof32.90(Table 4 and Figure 4E)ofDENV-1,DENV-2,DENV-4,YFV andZIKVcDNAdidnotgenerateanysignificant increaseinfluorescence,indicatinganabsenceofthespecificDENV-3target.Likewise,intheamplificationperformedtodetectDENV-4,theDENV-4cDNAshowedasignificantincreaseinthefluorescenceofSYBRGreenwithameanCTof31.28(Table 4 and Figure 4F).Similarly,theamplificationofDENV-1,DENV-2,DENV-3,YFVandZIKVcDNAdidnotgenerateanysignificantincreaseinfluorescence,indicatingtheabsenceofthespecificDENV-4target.

Thedissociationcurvesshowedonlyasinglepeakatthemeltingtemperature (Tm=80.0°C) expected for the DENV-1 amplicon

Dilution positive Viral§

Ct mean of the triplicate reaction indicating the serotype and pair of primers used No. of copies*DENV-1 DENV-2 DENV-3 DENV-4

STD1-rTS1 STD2-mTS2 STD3-TS3 STD4-rTS41:10 11.03 14.87 11.58 14.87 106

1:100 14.14 16.94 14.86 18.02 105

1:1.000 17.64 18.09 18.21 21.09 104

1:10.000 21.19 21.28 22.23 24.85 103

1:100.000 24.31 24.39 25.49 28.04 102

1:1.000.000 27.78 28.99 29.46 32.00 101

1:10.000.000 30.76 32.79 32.75 35.72 100

H2O N/A N/A N/A N/A N/A*Onlyvalues(log)ofdilutionarepresented,becauseonumbercopyiscalculatedfromsizeinnucleotidebasePairs.§Theviralplasmidwasusedasatemplate

Table 2A PerformancefortheSYBRGreenassayusingDENV1-4plasmidfragmentandtheserotype-specificprimers.

Dilution of the viral sampleCt mean of the triplicate reaction indicating the serotype and pair of primers used

DENV-1 DENV-2 DENV-3 DENV-4STD1/rTS1 STD2/mTS2 STD3/TS3 STD4/rTS4

1:10 7.36 14.99 10.95 11.971:100 10.96 16.98 13.93 13.951:1.000 14.96 19.96 17.94 17.961:10.000 17.97 23.96 21.94 21.951:100.000 20.94 27.97 24.92 24.951:1.000.000 24.95 31.98 28.92 27.94

H2O N/A N/A N/A N/A

Table 2B PerformancefortheSYBRGreenassayusingDENV1-4cDNAandtheserotype-specificprimers.

Viral samples Coefficient of angular PCR efficiency values (%)DENV-1cDNA 0.996 92.6

Plasmid 0.999 100.0DENV-2cDNA 0.987 93.7



Plasmid 0.998 94.0

Table 3 Coefficient of angular and PCR efficiency values for the SYBRgreen assay using the primers-specific in the viral cDNA samples andplasmid viral.

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Figure 4 TheamplificationprofilesandthedissociationcurvesofDENV1-4cDNAusing theSYBRGreenPCR assay. Theamplificationprofileisshownwheretheamplificationplotcrossesthethresholdlineforeachamplicon.Themeltingtemperature(Tm)ofeachampliconisshownalongsideitsdissociationcurve.

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using the DENV-1 cDNA, but not in the other cDNA samples(Figure 4B). Additionally, only a single peak at the meltingtemperature (Tm=79.8°C)was found for the DENV-2 ampliconusingDENV-2cDNA,butnot intheothercDNAsamples(Figure 4D). Similarly, only a single peak at the melting temperature(Tm=79.5°C)wasfoundfortheDENV-3ampliconusingDENV-3cDNA,butnopeakswerepresentinthenegativesamples(Figure 4F).Asinglepeakwasobservedatthemeltingtemperature(Tm=80.3°C)asexpectedfortheDENV-4ampliconusingintheDENV-4cDNA,butnopeakswerepresentinthenegativecDNAsamples(Figure 4H).

Discussion Currently, commercial kits to DENV detection has been used,CDC DENV-1-4-RT-PCR Assay for Detection and SerotypeIdentificationofDengueViruskit(CDCDENV1-4RT-PCRassaykit)todetectDENVwasbasedonthearticleofJohnsonetal.[26].Theseauthorsuse for serotype-specificdetectionofdengue inafourplexReal-TimeReverseTranscriptasePCRAssay.TheuseCDCDENV-1-4-RT-PCRkithassomegoodadvantage,suchasfourserotypes can be identified, simultaneously, using 4 differentprobesandeachserotypecanbealsoidentifiedseparatelyusingthe same probe. But some disadvantages are also observed:first, the cost of the kit is elevated; it needs 3 oligonucleotideprimers and 2 of them are fluorogenic probes used in theserotype-specific DENV by RT-qPCR assay. The oligonucleotideprimersto identifyDENV1-4weredesignedindifferentregionsofthegenomeofeachDENV,thisfactmakesimpossibletheuseofPCR-nested technique.Furthermore, theDENVserotyping isdoneusingInvitrogen2XPCRMasterMixandSuperscriptIIIRT/PlatinumTaqenzymemix,withelevatedcost.IntheCDCDENV1-4RT-PCRassaykitinstructionhighlightsthesensitivityofthetestisanother important issue. Itwasobservedthatonlyelevatedplaque-forming unit (PFU), approximately 1× 103- 107, couldbecorrelatedwithCTvalues.Although,CTvaluecorrelationto103pfu/mLofserumisat36.36,36.58and36.68forDENV1,3and4, respectively.According to Johnsonet al. [27], a sampledeterminedempiricallytobenegative if theCTvaluewas>36,allowing theconclusion that theCDCDENV-1-4-RT-PCRkit testhaslowsensitivity.

Najioullah et al. [28] evaluated four commercial RT-qPCR kitsand compared to Lanciotti's gold-standard protocol [18]. All

commercialkitsuse6-carboxyfluorescein(FAM)probeinTaqMansystem.ForthreeofthemnamelyGeno-sen's,Liferiver,Realstar,thetargetregionofvirusisnotspecifiedandthesekitscannotbeusedforDENVserotyping.OnlyfortheSimplexakit,thetargetregionofDENV1-4isknownandcanbeusedforserotyping.Theauthorsutilizedapanelwith162laboratory-confirmedsamples:46DENV-1,37DENV-2,33DENV-3and46DENV-4.Theresultsshowed thatnocommercialRT-qPCRkits testedshowed100%sensitivityinthedetectionofallDENV,Geno-sen'ssensitivityis85.2%,Realstar83.3%andSimplexa93.2%.Ontheotherhand,Lanciotti's gold-standard protocol is indicated for DENV1-4serotypingwithprimersdesignedinC-prMregion[29]redesignedprimerstodetectDENV1-4inthesameregionandshowedthatC-prMregionisindeedmoresensitive(100%)thantheNS5(91%)orthe3’NC(91%)protocolstodetectDENV.

Inrecentyears,quantitativePCRbasedontheTaqMansystemorSYBRGreenassayshavebeenusedtodetectseveralRNAvirusesin infectedplants [30,31], animals [3,7,16,22,27,32,33],humanfluids[1,2,4,5,8,10,14,19,20,34,35]amongotherapplications.

Quantitative PCRmethods such as the SYBR Green assay andTaqMan detection system have been traditionally used forDENV serotyping to provide a compatible dynamic range andsensitivity.SYBRGreendetectionwasmoreaccurateandyieldedamore linear decay graph than the TaqMandetection system[36,37]. In addition to the sensitivity and specificity of thetechniquedescribedinthiswork,SYBRGreenPCRassayisveryfastandrobustinnature.The96-wellplateinthisworkcanbeusedtoanalyzebetween6-10sampleswith2or3replicates ifDENV1-4serotypesweretestedonthesameplate,usingpositiveandnegativecontrols. It takesabout2hours toperforma96-well plate. After amplification, data analysis may take severalminutes. In contrast, the TaqMan system detection can beusedwithmultiplefluorogenicprobestodetectmorethanoneDENVserotypeinthesamereaction [15].Although,theTaqManassay ismuchmoreexpensive than theSYBRGreen trial [6,7],whichcorroborates totheuseofSYBRGreenfortestingassaysin research laboratories and serological diagnosis in developing countries.

The DENV serotyping protocol adopted by Chien et al. [8]includedanexpensiveapproach,aone-stepRT-PCRmastermixkit and a second reactionwith aHotStarTaqmastermix, both

Sample Viral*

Ct mean of the triplicate reaction indicating the serotype of DENVs and other Flaviviruses and pair of primers used

DENV-1 DENV-2 DENV-3 DENV-4STD1-rTS1 STD2-mTS2 STD3-TS3 STD4-rTS4

DENV-1 28.18 N/A N/A N/ADENV-2 N/A 32.79 N/A N/ADENV-3 N/A N/A 32.90 N/ADENV-4 N/A N/A N/A 31.28ZIKV N/A N/A N/A N/AYFV N/A N/A N/A N/AH2O N/A N/A N/A N/A

*cDNAofDENV1-4fromanewlypassagedcellcultureswasusedwiththeirspecificandnon-specificsetsofprimersfordetection.

Table 4 Performanceofspecificityoftheserotype-specificprimersintheSYBRGreenassayusingDengueVirus1-4andotherFlaviviruses.

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kitssuppliedbyQIAGEN.ReductionsintheoperationalcostsbyusingacheaperDNApolymeraseenzymeandanincreaseintheassaysensitivity todetectDENV1-4werethemainmotivationsfor redesigning ormodifying the previously established C-prMprimersets[6,18].

In this study,we also chose the C-prM gene region and novelprimersweredesignedtobecompatibletocheaperpolymeraseenzymes for theuse in routineof laboratory.WemodifiedtheampliconsfromanexistingprotocolfortheC-prMjunction[6],designing5newforwardprimerstoformsetsforDENVserotyping.Thisnewlyestablishedprotocolusesasemi-quantitativenestedPCRdetectionmethodandaquantitativePCRSYBRGreenassay.

TodetectallDENVs,weperformeda semi-quantitativenestedPCRtechnique(Figure 2).WereplacedtheforwardprimermD1[6]withtheSTDprimerinthefirstreactionoftheassay.Inthesecondreaction,wealsoreplacedthemD1[6]forwardconsensusprimerwiththemorespecificforwardprimersSTD1,STD2,STD3andSTD4,oneforeachDENVsequence,creatinganestedPCRassayusingPhusionDNApolymerase,acheaperenzymethantheenzymesdescribedintheliterature[6,18,33],therebyincreasingtheefficiencyand reducing thecostsof theprotocol forDENVserotyping.

Toobtainaccurateandreproducibleresultswiththenewspecificforwardprimers (STD1,STD2,STD3andSTD4)and the reverseprimersadoptedbyChienetal.[5]todetectDENV1-4,theSYBRGreen PCR efficiencywas determined, its range should be 90-110%indicatingadoublingoftheampliconateachcycle.Table3showsthatthePCRefficiencyvalueswiththenewprimersets(STD1/rTS1; STD2/mTS2; STD3/TS3 and STD4/rTS4) using bothviralplasmidandcDNAastemplateswerehigherthanthevaluesfound inthe literature[6],except forDENV2cDNA,confirmingtheappropriatedesignofthenewprimersforDENVserotyping.

The specificity of the new primer sets to detect DENV1-4wasdeterminedthroughananalysisofmeltingtemperaturecurvespost-amplificationusingaSYBRGreenassay.Adissociationcurvewithasinglepeakatthetemperatureexpectedforeachampliconindicatedaspecificamplification.InFigure4,thepresenceofasinglepeakcanbeobservedforeachDENV1-4cDNAtested,withnopeaksappearingusingcDNAsfromotherscontrolflaviviruses. The specificity of the DENV primer set was also confirmed byamplification profiles of themelting dissociation curves of thetarget products (Figure 4). In a SYBR Green PCR, a sample isconsidered positive when the amplification plot crosses thethreshold line. In theexampleof (Figure 4A), theamplificationprofileofthesample infectedwithDENV-1canbeobservedtoexceed the threshold line, whereas the amplification profilesof the other viral samples do not reach this threshold line. This parameter represents the baseline of fluorescence detection,that is, the minimum number of cycles for amplification. Thisvalue isdeterminedby thesoftwareanalysis systemused.Thepointatwhichthesamplefluorescencesignalishigherthanthefluorescencedetection/quantificationlimit,indicatesthenumberofcyclesrequiredtoinitiatetheamplificationofthegenetargetsequencepresentintheDNAofeachsampletobedetermined.Thisvalue iscalledCycleThreshold (Ct)andallowstherelative

DNAquantificationofeachsampleafterbeingcorrectedbytheCtof thegeneendogenouscontroland/or thesamplecontrol.TheCt isproportionaltothe logarithmofthe initialamountofexpressionofthetargetgeneinagivensample.ThesmallertheinitialnumberofCtobtainedfromthetargetgeneinthesamplecomparedtoanothergeneindicatesthegreaterexpression.

Thereal-timeRT-PCRdescribedinthisstudyishighlysensitive,andabletodetectuptoalittleaboveonecopyofDENVgenome(Figure 4).InSYBRGreenPCR,amaximumof40cycles(CT=40)is required to detect a single copy of a viral genome (CFX96Touch Real-Time PCR Detection System, Bio-Rad Laboratories,Hercules,California,USA).AlinearrelationshipbetweenplasmidDNAcopiesandCTvaluesfrom106upto3copiesofDENVcouldbe observed. Due to high sensitivity of SYBR Green PCR, it issusceptibletoPCRcarryoverorothercontamination.Therefore,good laboratory practices should be followed very strictly topreventanypotentialcontaminationthatmaygivefalsepositiveresult.Inaddition,anynegativeresultaswellassampleswithCtvaluescloseto36shouldbetestedatleasttwiceforconfirmation.TheSYBRGreenPCRprotocoldescribedherewasnotonlyveryhighlyefficientandsensitive,butalsoshowedspecificityforthedetectionofDENV1-4,betterthandescribedbyChienetal.[22].Its specificity was determined bymonitoring the amplificationprofileonthemeltingdissociationcurveofthetargetproducts.InSYBRGreenPCRreaction,asampleisconsideredpositivewhenthe amplification plot crosses the linear threshold line. As wecansee,intheexample(Figure 4A)theamplificationplotofthesample infectedwithDENV-1exceedsthethreshold line inthecycle(28.18),whereasthegraphofamplificationofthenegativesampledoesnot.ThedissociationcurveprofiledependsonCGbases composition and amplified sequence length, the non-specific productmust be differentiated by examination of thedissociationcurve.ThefragmentamplifiedforDENV-1produceda specific dissociation curve exhibiting a single peak at 80.0°C,whichisexpectedfortheDENV-1specificamplificationproduct.Aiming to determine the specificity, quality and efficiency ofthe reaction, cDNAs frombothviral sampleswereamplified inparallel foreachtargetvirus.Usingthismethod,wewereabletodiscriminateeach targetby itsdissociation curve, accordingto the predictedmelting temperature (Tm for DENV-2 was of79.8°C,TmforDENV-3wasof79.5°CandTmforDENV-4wasof80.3°C,(Figure 4D, F and H).

Theprotocolsestablishedinthisworkusingthesemi-quantitativenesteddetectionmethodandthequantitativePCRSYBRGreenassaywiththenewprimersets(STD1/rTS1;STD2/mTS2;STD3/TS3andSTD4/rTS4)canbeused,independentlyorconcomitantly,fortheidentificationoftheDENVserotypes. Therefore, this present manuscriptisanupgradeoftheChienetal.[22]paper.

ConclusionThe semi-quantitative nested PCR and SYBR Green PCRtechniquesdescribedaboveareimportanttoolsinthedetectionand quantification of the four serotypes of Dengue virus andcanbeused fordetection inmosquitovectors,serumorotherhumanfluids,aswellasintestsfordose-responsesofantivirals

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incellculturesorthedeterminationofviralloadsinothertypesof studies. The SYBRGreenquantitativemethod is fast, highlysensitive, robust, low cost, and easy to reproduce. It is alsoapplicable to routine high-performance testing, making it apowerfultoolsuitableforthirdworldcountriestryingtopreventthe propagation of DENV by monitoring the epidemiologicalmovementbetweencountriesandcontinents.ThereisagrowingandurgentneedtodevelopmoreaccessibleandhighlysensitiveDENVdetectionmethodssuchasthemethodologydescribedinthisstudy.

Compliance with ethical standards Conflict of interestTheauthorsdeclarethattheyhavenoconflictofinterest.

Ethical approvalThisarticledoesnotcontainanystudieswithhumanparticipantsoranimalsperformedbyanyoftheauthors.

Author ContributionsT.S.S.,M.R.S.,D.F.FandM.F.Mdesignedtheexperiments.T.S.S.,T.E.S.GandD.F.Fpreparationofviruses.T.S.S,T.E.S.G.,D.F.S.S,S.G.L., T.A.F. performed PCR assays. T.S.S., T.E.S.G., E.S.L.A.,A.C.A.M., M.R.S. and M.F.M performed database searches,sequenceanddata analyses.T.S.S.,M.R.S.andMFM.wrotethemainmanuscript.Allauthorsreviewedthemanuscript.

Acknowledgements This studywas supportedfinancially by theConselhoNacionalde Desenvolvimento Científico e Tecnológico (CNPq), InstitutoNacional de Ciência e Tecnologia em Entomologia Molecular(INCT-EM/CNPq), Fundação Carlos Chagas Filho de Amparo àPesquisadoEstadodoRiodeJaneiro(FAPERJ)andCoordenaçãodeAperfeiçoamentodePessoaldeNívelSuperior (CAPES).The authors are grateful to Dr. Rafael DiasMesquita for providingassistance toaccomplishthiswork.

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