In-house quantitative real-time PCR for the diagnosis of hepatitis B virus and hepatitis C virus infections

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Genetics

and

Molecular

Microbiology

In-house

quantitative

real-time

PCR

for

the

diagnosis

of

hepatitis

B

virus

and

hepatitis

C

virus

infections

Danielle

Alves

Gomes

Zauli

a,∗

_,

_Carla

_Lisandre

_Paula

_de

_Menezes

b

_,

Cristiane

Lommez

de

Oliveira

a

_,

_Elvis

_Cristian

_Cueva

_Mateo

a

_,

Alessandro

Clayton

de

Souza

Ferreira

a

a_{InstitutoHermesPardini,DepartamentodePesquisaeDesenvolvimento,Vespasiano,MG,Brazil} b_{LinhagenProdutosemBiotecnologiaLtda,BeloHorizonte,MinasGerais,Brazil}

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Articlehistory:

Received20July2015 Accepted20February2016 Availableonline27July2016 AssociateEditor:MaurícioLacerda Nogueira Keywords: qPCR HepatitisB HepatitisC

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b

s

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Thequantificationofviralnucleicacidsinserumbyreal-timePCRplaysanimportantrole indiagnosinghepatitisBvirusandhepatitisCvirusinfection.Inthisstudy,wedeveloped anassayusingspecificprimersandprobestoquantifyhepatitisBvirusDNAorhepatitisC virusRNAinserumfrominfectedpatients.Forstandardizationandvalidationoftheassay, aninternationalpanelofhepatitisBvirus/hepatitisCvirusandstandardplasmidswas used.Acorrelationcoefficientof0.983and0.963forhepatitisBvirusandhepatitisCvirus, respectively,wasobtainedbasedoncyclethresholdvaluesandconcentrationsofDNAor RNA.Thestandardcurveshowedalinearrelationshipfrom19IU/mLto1.9×109_IU/mLof

serum,withacoefficientofdetermination(r2_{)of0.99.Inserafrompatientsinfectedwith}

hepatitisBvirusorhepatitisCvirusviralloads(19IU/mLand1.9×109_{IU/mL),wequantified}

viralloadswithadetectionlimitof1.9×102_{IU/mL.Thereal-timequantitativePCRassay}

developedinthisstudyprovidesanidealsystemforroutinediagnosisandconfirmationof indeterminateserologicalresults,especiallyinimmunosuppressedpatients.

©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Acute and chronic infections with hepatitis B virus (HBV) orhepatitisC virus (HCV)leadtosignificant mortalityand areamajorpublichealthproblemworldwide.According to

∗ _{Correspondingauthorat:DepartamentodePesquisaeDesenvolvimento,InstitutoHermesPardini,BeloHorizonte,Av.Nac¸ões,2448,}

PortariaA,DistritoIndustrial33200-000,BeloHorizonte,MG,Brazil. E-mail:[email protected](D.A.Zauli).

theWorldHealthOrganization(WHO),130–150million peo-pleareinfectedwithHCVworldwideand240millionpeople arechronicallyinfectedwithHBV.Anestimated30%ofpeople infecteddevelopliver cirrhosisand/orhepatocellular carci-noma(HCC).1–3

http://dx.doi.org/10.1016/j.bjm.2016.07.008

1517-8382/©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

HBVandHCVinfectionisusuallydiagnosedbythe detec-tionofanti-HBV/oranti-HCVantibodiesinapatient’sserum thatreacttorecombinantHBVorHCVproteinsinanenzyme immunoassay or chemiluminescence immunoassay. How-ever,thesemarkershavelimitationsthatreducediagnostic accuracy.Inordertoovercometheseproblems,severalassays havebeen developedinthelastfewyearstodetectnucleic acidsofhepatitis-causingviruses.4–8

DeterminingHBV DNAorHCV RNAlevelsinserum has becomeanimportanttooltoidentifyindividualswithhigh viral loads,whichmay suggest high infectivity,to monitor diseaseprogressionandtheefficacyofantiviraltherapies,to detectdrugresistant mutants,and toidentifyrelapse after thediscontinuationofanantiviraltherapy.9–12_{Alimitationin}

hepatitisvirusresearchisthelackofasensitiveandhighly specifictesttomeasureviralloadsinplasmaorserum. Pre-viousstudieshaveusedreal-timePCR-basedapproaches to determineviralloadsininfectedpatients.Withhigh sensi-tivity and specificity, broaddetection capability, simplicity, andreproducibility, thistechniqueisparticularlyuseful for theanalysisofalargenumberofspecimensandtomeasure viralload.13–16 _{Inthe present study,we developeda}

sensi-tivemethodtoquantifyHBVDNAandHCVRNAinserafrom infectedpatientsbyquantitativereal-timePCR(qPCR)using specificprimersandTaqManminorgroovebindingfluorescent probetechnology.

Materials

and

methods

Patientsandserumsamples

Atotal of116 serum sampleswere obtainedfrompatients infectedwithHBV orHCV.Thisstudywasapprovedbythe EthicsCommitteeofUniversidadeFederaldeJuizdeFora (pro-tocol042/2010).

Selectionanddesignofprimersandprobesfor

quantitativereal-timePCR

Thesequencesoftheprimerand probesusedinthisstudy are listedin Table1.Theprimers were selectedfrom pub-lishedliterature,andtheprobesweredesignedusingPrimer Express Software (Applied Biosystems, Foster, CA, USA). Programs, including AmplifX (CNRS, Aix-Marseille Univer-sité,http://crn2m.univ-mrs.fr/pub/amplifx-dist),wereusedto

predictthebehavioroftheseprimers.Theexpected hybridiza-tionandspecificityofprimer–probesetstargetingHBVDNA andHCVRNAweredeterminedbyinsilicoanalysisusingthe BLASTprogram(http://ncbi.nlm.nih.gov/BLAST).Thereporter dyeFAMwasattachedtothe5 endsofprobes,anda non-fluorescentquencher(NFQ)and minorgroovebinder(MGB) were attached tothe 3 ends. All primersand probes were synthesizedbyIntegratedDNATechnologies.

Extractionofnucleicacids

Nucleic acids were extracted from 200␮L of serum using a QIAamp MinElute Virus Spin kit (Thermo Fisher Scien-tific, Waltham, MA, USA) according to the manufacturer’s suggested protocol. The concentration and quality of the extracted DNA and RNA were assessed by Nanovue spec-trophotometry(ThermoFisherScientific,Waltham,MA,USA) and by amplification of a fragment of the gene coding for␤-actin(internalcontrol) (GenBank No.AY5827991).The extractedDNAandRNAwerestoredat−80◦_Cuntiluse.

cDNAsynthesis

ViralRNAwasreversetranscribedusingrandomprimersanda HighCapacitycDNAReverseTranscriptionKit(ThermoFisher Scientific,Waltham,MA, USA)followingthemanufacturer’s suggestedprotocol.TheresultingcDNAwasstoredat temper-ature−20◦_Cuntiluse.

ConventionalPCR

ConventionalPCRwasusedtoevaluatethesizesofamplified products.Theamplificationconditionswerethefollowing:a 10-␮Lreactionmixturecontaining50–100ng/LtemplateDNA, 0.1␮mol/Lprimermixture,1␮mol/Ldeoxynucleoside triphos-phates,1␮Lof10×buffer,2mmol/LMgCl2,and0.05␮LGold

StarTaqpolymerase,withcyclingconditionsthatincludedan initialdenaturationandpolymeraseactivationstepfor10min at 95◦C,followed by 35 cycles ofdenaturation at95◦C for 1min,primerannealingat60◦Cfor1min,extensionat72◦C for2min,andthenafinalextensionstepof10minat72◦C. AllPCRproductswerevisualizedfollowing6%polyacrylamide gelelectrophoresisandstainingofgelswithsilvernitrateas describedbyRef.19.

Table1–Primersandprobesusedinthisstudy.

Virus Gene Sequencesofprimersandprobes(5–3) Ampliconsize(basepairs) Reference HBV Sgene a_{FW:CCTGGYTATCGYTGGATGTGT}

b_{RV:GGACAVACGKGCAACATACCTT}

Probe:6FAMCTATGCCTCATCTTCTTMGBNFQ

116 17

HCV Non-codingregion FW:AGCGTCTAGCCATGGCGTT RV:GCAAGCACCCTATCAGGCAGT

Probe:FAMTCTGCGGAACCGGTGAGTMGBNFQ

238 18

a _{Forwardprimer.} b _{Reverseprimer.}

OptimizationoftheqPCRassay

The qPCR assay was optimized following the MIQE guidelines.20 _{Primer and probe matrix experiments were}

conductedbyselecting, foreach gene,the primer concen-trationthat resultedinthe lowestcycle threshold(Ct) and the highest Rn using a fixed amount of target template. Reactionswithdifferentconcentrationsofprimersandprobes wereperformedintotalvolumesof25␮Land12.5␮L,of Taq-ManUniversalMasterMixbuffer(AppliedBiosystems,Foster CA,USA);senseprimerandantisenseprimerconcentrations of50nM,300nM,and900nMeach;probesatconcentrations of80nM, 125nM, 150nM, and 250nM; and 1␮LofDNA or cDNA.The reaction wasperformed with a7500 Real-Time PCRSystem (ThermoFisher Scientific, Waltham, MA, USA) usinguniversalconditions:50◦Cfor2min,95◦Cfor10min,45 cyclesof95◦Cfor15s,and60◦Cfor1min.Forthesereactions, an OptiQuant HBV/HCV Quantification Panel (AcroMetrix, Benicia,CA,USA)wasusedinthefollowingconcentrations expressedininternationalunits/mL(IU/mL):HBV7orHCV7 (5,000,000IU/mL)andHBV2orHCV2(50IU/mL).

Validationoftheassaywithinternationalstandards

Forvalidationoftheassay,theinternationalpanelcitedabove wasusedinqPCRreactionstoconstructstandardcurvesof HBVDNAorHCVRNAinthefollowingconcentrations:HBV 7orHCV7(5,000,000IU/mL),HCV6orHBV6(500,000IU/mL), HBV5orHCV5(50,000IU/mL),HBV4orHCV4(5000IU/mL), HBV3orHCV3(500IU/mL),andHBV2orHCV2(50IU/mL). Thereactionswereperformedina7500Real-TimePCR Sys-tem(ThermoFisherScientific,Waltham,MA,USA)usingthe TaqMandetectionsystem(ThermoFisherScientific,Waltham, MA, USA)with 12.5␮L ofTaqMan Universal Master Buffer, predeterminedconcentrationsoftheprimer–probesetscited above,and50-100ngofDNAorcDNA,foratotalfinal vol-umeof25␮Lper reaction. Allreactions wereperformed in duplicateusinguniversalconditions:50◦Cfor2min,95◦Cfor 10min,45cyclesof95◦Cfor15s,and60◦Cfor1min.Results wereanalyzedusing7500FastSoftwarev.2.1(Life Technolo-gies,Carlsbad,CA,USA)andexpressedinIU/mL.Thebaseline and thresholdvalues wereautomatically adjustedforeach test.

Quantificationofviralloads

Toquantifytheviralloads,standardcurveswereconstructed fromadilutionseriesofclonedplasmidscontaininginsertsin theconservedregionofeachvirusininitialconcentrationsof 5.1×109_{IU/mLand4.9×10}9_{IU/mLforHBVandHCV,}

respec-tively,withatotalofeightpointsineachcurve.Thefragments wereamplifiedbyconventionalPCRandclonedusingaTOPO TACloningKit(Invitrogen,California,CA,USA)accordingto manufacturer’sinstructions.Thecloningandtransformations wereperformedusingaCloneJETPCRCloningKitand Trans-formAidBacterialTransformation Kit, respectively(Thermo FisherScientific,Waltham,MA,USA)followingthe manufac-turer’sprotocol.Theconcentrationofrecombinantplasmids wasdeterminedusingaQubit2.0Fluorometer(ThermoFisher

Scientific,Waltham,MA,USA),andreactionswereperformed in a7500Real-TimePCR System(ThermoFisher Scientific, Waltham,MA,USA)usingtheTaqMandetectionsystem(Life Technologies,Carlsbad,CA,USA).Assaysconsistedof12.5␮L ofTaqManUniversalMasterBuffer,predetermined concentra-tionsofprimersandprobes,and50–100ngofDNAorcDNA, forafinalvolumeof25␮Lperreaction.Allreactionswere per-formedinduplicate.Resultswere analyzedusing7500Fast Softwarev.2.1(ThermoFisherScientific,Waltham,MA,USA) andexpressedinIU/mLofthetargettemplate.Thebaseline and thresholdvalueswere automaticallyadjustedforeach test.Therelativeefficiencyofamplificationwasassessedby theslopeofalinearregressionofCtvaluesagainst concen-trationsofDNAorcDNA.Theefficiencyofthereactionwas determinedaccordingtothefollowingformula20_:

E=



10



₋₁ slope



−1



×100.

Validationoftheassaywithserumsamples

To validatethis assay, serum sampleswere collected from patientsinfected,eitheracutelyorchronically,withHBV or HCVandmaintainedat−20◦_{Cuntiluse.Theinclusion}

crite-rionwasthepresenceofHBVorHCVinfection.Theexclusion criterionwastheabsenceofHBVorHCVinfection.Thisset of49HBVand67HCVpositiveserawereusedtovalidatethe qPCR assay.For theqPCRreactions, adilutionseriesofthe standardplasmidwasusedasthestandardcurve.

Limitofdetection(LOD)

The LOD ofthe assay was determined byserially diluting the standardplasmid(1.9×1010_–0.19×100_IU/mL)and

iden-tifyingthelowestconcentrationofeachcloneyieldinga90% orgreaterdetectionrate.

Results

ConventionalPCR

ThesizesoftheproductsamplifiedbyconventionalPCRare presentedinFig.1.Productswereoftheexpectedsize, indi-catingamplificationofthetargettemplate.

StandardizationofqPCR

InordertostandardizetheqPCRassayforquantificationof HBVorHCV,differentprimerandprobeconcentrationswere usedtodeterminetheoptimaldetectionconditions.Table2

showstheCtvaluesandslopeofthelinearregressionfrom reactionsattheseconcentrations.UsingtheHBV7orHCV7 controlsastemplates,amplificationwasdetectedinall com-binations ofprimersets.For the HBV 2orHCV 2controls, amplificationwasdetectedonlyatforwardandreverseprimer concentrationsof50nMand300nM,respectively.Wefoundno differenceinassayperformanceatthevariousprobe concen-trations,andafinalprobeconcentrationof125nMwasused inallqPCRreactions.

250 MW HCV HBV 200 150 100 50

Fig.1–Gelelectrophoresis(6%polyacrylamide)ofproducts amplifiedfromHBVandHCVcodinggenesbyconventional PCR.MW,molecularweightstandard(50bp).

ValidationoftheqPCRassaywithinternationalstandards

To validate the assay for quantitationof HBV and HCV, a standardcurvewasconstructedusingHBVDNAorHCVRNA from an international panel. The amplification plots and standardcurvesofbothvirusesareshowninFig.2. Regres-sionsoftheCtvaluesandconcentrationsofHBVDNAorHCV cDNAshowedcorrelationcoefficientsof0.983and0.963for HBVandHCV,respectively.Theslopesofthestandardcurves forHBVandHCVwere−3.438and−2.898,respectively.

Quantificationofviralloads

In order to obtain a standard curve for absolute quantita-tionofHBVDNAandHCVRNA,10-folddilutionsofstandard plasmidswereusedastemplates.Theamplificationplotand standardcurve(Fig.3)thatwere generatedshowedalinear relationshipfrom5.1×102to5.1×109IU/mLand4.9×102to 4.9×109_{IU/mLforHBVandHCV,respectively.Theslope}

val-ueswere−3.583 and−3.263forHBVandHCV,respectively. Linearregressionanalysisyieldedacoefficientof determina-tion(r2_{)of0.99andefficienciesof90–100%forbothHBVand}

HCV.

Validationoftheassaywithserumsamples

Atotalof49serumsamplesfrompatientsinfectedwithHBV and 67serum samplesofpatientsinfectedwith HCVwere analyzedbyqPCR.Inallclinicalsamples,it waspossibleto

Table2–Results(Ctvaluesandslope)fromtheqPCR reactionsusingdifferentconcentrationsofprimer–probe setstargetingconservedHBVandHCVgenes.

International standard Primer concentration FW/RV Ctvalue Slope value HBV7a _{50nM/50nM 28.86785507 −1.0434} 50nM/300nM 26.90498924 50nM/900nM 26.2933445 300nM/50nM 25.48120117 300nM/300nM 23.62826729 300nM/900nM 23.69248581 HBV2b _{50nM/50nM 37.58005905 1.4634} 50nM/300nM 39.04344177 50nM/900nM Undetermined 300nM/50nM Undetermined 300nM/300nM Undetermined 300nM/900nM Undetermined HCV7a _{50nM/50nM 29.46348 0.7627} 50nM/300nM 29.3045578 50nM/900nM 28.48422432 300nM/50nM 28.22242737 300nM/300nM 31.08761597 300nM/900nM 33.78520966 HCV2b _{50nM/50nM Undetermined Undetermined} 50nM/300nM 36.86901855 50nM/900nM Undetermined 300nM/50nM Undetermined 300nM/300nM Undetermined 300nM/900nM Undetermined

a _{Viralload:5,000,000IU/mL(HBV7/HCV7).} b _{Viralload:50IU/mL(HBV2/HCV2).}

quantifytheviralloadandoptimizetheassayforacceptable sensitivity.

LODoftheassay

Adilutionseriesofstandardplasmidsforeachvirus,ranging from1.9×1010_to0.19×100_{IU/mL,wasusedtodeterminethe}

LODoftheassay.The90%LODfortheassaywasfoundtobe 1.9×102_{IU/mLforeachvirus.}

Discussion

Inthisstudy,wedevelopedanin-houseqPCR-basedassayfor the quantificationofHBV DNAand HCVRNA insera from infected patients. Theassay, proposedforuse in diagnosis of HBV and HCV, amplified conserved regions of the HBV andHCVgenomesandwasstandardizedandvalidatedusing different concentrations of primer–probe sets and interna-tionalnucleicacidstandards.ViralDNAorRNA,isolatedby acommonlyusednucleicacidextractionkit,couldbeusedto quantifyvirusinaninitialsamplevolumeofonly200␮L.

The primers used for HBV detection are specific for a highlyconservedregionoftheSgene.17_{All HBVgenotypes}

foundinBrazilianpatientsanddescribedinpreviousstudies weredetectedwiththesameprimersusedinthisstudy.17,21

Theprimers selectedforHCVtarget the conserved5 non-translatedregionoftheviralRNA.Accordingtotheliterature, allgenotypesofHCVcanbedetectedwiththeseprimers.18

10 0.287931 0.238246 40.0 37.5 35.0 32.5 30.0 27.5 25.0 22.5 40.0 37.5 35.0 32.5 30.0 27.5 25.0 20.0 22.5 10 2030 100200 1000 10 000 100 000 1 000 000 10 000 000 1 000 000 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 2 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 24 36 38 40 42 44 46 48 50 _{0.10.2 1 2345 10 2030 100 200 1000 10 000 10 0000 1 000 000 10 000 000 1 000 000} 4 6 8 10 12 14 16 18 20 22 24 26 Cycle Quantity Cycle Quantity Standard curve Standard curve Amplification plot Amplification plot

C

D

A

B

28 30 32 34 36 38 40 42 44 46 48 50 Δ Rn Δ Rn CT CT

Fig.2–AmplificationplotandstandardcurveofHBV(AandB,respectively)andHCV(CandD,respectively)obtainedusing theInternationalOptiQuantHBV/HCVQuantificationPanel(AcroMetrix,Benicia,CA,USA)tovalidatetheqPCRassay.The resultsareexpressedininternationalunits/mL(IU/mL).

10 37.5 40 35 30 25 20 15 10 5 102030 100 200 1.000 10 000 100 000 1 000 000 10 000 000 100 000 000 1 000 000 35.0 32.5 30.0 27.5 25.0 22.5 20.0 17.5 15.0 12.5 10.0 7.5 1 2345 10 2030 100200 1.000 10 000 Quantity Standard curve Amplification plot

A

B

C

D

0.019022 0.046841 Standard curve Amplification plot Cycle Quantity Cycle 100 000 1 000 000 10 000 000 1 000 000 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Δ Rn 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 Δ Rn CT CT

Fig.3–AmplificationplotsandstandardcurvespreparedusingstandardplasmidsforquantificationofHBV(AandB, respectively)andHCV(CandD,respectively)viralloads.ThedilutionseriesofthestandardplasmidsofHBVDNAandHCV RNAbeganat5.1×109_{IU/mLand4.9×10}9_{IU/mLforHBVandHCV,respectively.TheresultsareexpressedinIU/mL.}

Moreover,the5nucleasefluorogenicprobeusedinthisstudy hasgreaterspecificityandsensitivityandabroaderdynamic rangeofdetectionthan assaysthat useDNA bindingdyes. Therefore,theassayshouldallowaccuratequantificationof allHBVandHCVgenotypes.

Inthisstudy,theqPCRassaydetectedloadsofHBVDNA rangingfrom5.1×109 _to5.1×102_{IU/mLandHCVRNA}

ran-gingfrom4.9×109_to4.9×102_{IU/mLinserum.Furthermore,}

theassay efficientlyquantified bothviruses (r2_{=0.99), with}

anadequateLOD(1.9×102_{IU/mL)foranin-houseqPCR.To}

demonstratetheeffectivenessoftheqPCRassayproposedin thisstudy,clinicalsamplesfrompatientsinfectedwithHBV orHCVweretested. Theresultssuggestthattheassaycan beusedtoquantifybothlowandhighviralloadsandthatits amplificationefficiencyisstableoverarangeofinputcopy numbers.

Itis importanttonote that comparisonof resultsfrom thisassaywiththose fromcurrentlyrecommendedclinical protocolsareneededbeforethisassaycanbeusedfor long-termmonitoringofHBVandHCVtreatments.Inconclusion, inthisstudywedevelopedanovelqPCRassay,basedonthe TaqManMGBsystem, thatisrapid,sensitive,andaccurate. Thisassay,validatedwithbothinternationalstandardsand clinicalsamples,providesanidealsystemforroutine diagno-sis,toconfirmindeterminateserologicalresults,especiallyin immunosuppressedpatients,andtodetectviremiapriorto seroconversion.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This study was supported by grants from the Conselho NacionaldoDesenvolvimentoCientíficoeTecnológico(CNPq) and Fundac¸ão de Amparo à Pesquisa do Estado de Minas Gerais(FAPEMIG).

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