Review
Mini
review:
Current
molecular
methods
for
the
detection
and
quantification
of
hepatitis
B
virus,
hepatitis
C
virus,
and
human
immunodeficiency
virus
type
1
Guilherme
Albertoni
a,b,*,
Manoel
Joa˜o
Batista
Castelo
Gira˜o
b,
Nestor
Schor
aaFederalUniversityofSa˜oPaulo(UNIFESP),DepartmentofMedicine,NephrologyDivision,RuaBotucatu740,Sa˜oPaulo,04023-900,SP,Brazil bColsan(Associac¸a˜oBeneficentedeColetadeSangue),Sa˜oPaulo,SP,Brazil
1. Introduction
Thequalityandsafetyofbloodproductsusedintransfusionsare majorpublichealthconcerns.Inadditiontogeneralqualitycontrol (QC),theintroductionofgoodmanufacturingpracticesandroutine screeningofbloodmaterialsandproducts,hasensuredconsistency, quality,andsafetyinthe increasedproductionanduseofblood productsinrecentdecades.Newlydevelopedserologicaltestsand nucleic acid tests (NATs) have markedly reduced the risk of transmissionofhumanimmunodeficiencyvirus(HIV),hepatitisC virus(HCV),andhepatitisBvirus(HBV).1Inthepastdecade,rapid andsensitivemoleculartechniquessuchasPCRhaverevolutionized thedetectionofavarietyofinfectiousviruses.2–4
About10yearsago,molecularmethodsachievedasignificant levelofthroughputandautomation,enablingtheirintroductionas anadditionaltoolinbloodscreening.Theaimofthesetestswas primarilytopreventdonationsmadeduringthewindow-period from being used in transfusions.5,6 An international survey7 reported findings from the use of NATs in 330 million blood donationsandshowedoverallinfectionratesof1:447000forHCV,
1:111000forHIV,and1:66000forHBV.Upuntil2011,veryfewof Brazil’sbloodbanks(correspondingtolessthan5%ofthenational blood supply) had voluntarily introducedNAT screening. More recently,anHCV/HIVNATkitwasdevelopedbyBio-Manguinhos/ Fiocruz(RiodeJaneiro,Brazil).Thistestisnowusedinsomeof Brazil’slargepublicbloodbanksandisexpectedtobeextendedto allpublicbloodbanks,whenthetestmaybecomemandatory.8
In this article,wedescribe twoof themostcommonlyused licensedmolecularmethodsforthedetectionandquantificationof HIV,HCV,andHBVinblood.Wecomparedthelowerlimitsofvirus detectionofeachmethodandevaluatedtheoverallperformance.
2. Nucleicacidtesting(NAT)
The World Health Organization (WHO) recommends that diagnosticdevicesare‘ASSURED’:Affordable,Sensitive,Specific, User-friendly,RapidandRobust,Equipment-free,and Deliver-abletoendusers,especiallywithregardtodevelopingcountries. Nucleic acid amplification techniques (NAATs), however, typicallyrequireasignificantinvestmentinequipment,training, and infrastructure. Nevertheless, the development of these moleculartechniquesasdiagnostictoolshasbecomeincreasingly important.9–13
ARTICLE INFO
Articlehistory: Received6March2014
Receivedinrevisedform1April2014 Accepted9April2014
CorrespondingEditor:LarryLutwick, Kalamazoo,Michigan,USA
Keywords: NATtesting HIV HCV HBV
SUMMARY
Thedetectionofacutehumanimmunodeficiencyvirus(HIV),hepatitisBvirus(HBV),andhepatitisC virus(HCV)infectionisvitalforcontrollingthespreadofHIV,HBV,andHCVtouninfectedindividuals. Consideringthattheseviruseshavehighreplicationratesandareundetectablebyserologicalmarkers, earlydetectionupon transmissionis crucial.Various nucleicacid assayshave been developedfor diagnosticsandtherapeuticmonitoringofinfections.Inthepastdecade,rapidandsensitivemolecular techniquessuchasPCRhaverevolutionizedthedetectionofavarietyofinfectiousviruses,includingHIV, HCV,andHBV.Here,wedescribetwoofthemostcommonlyusedlicensedmethodsforthedetectionand quantificationofHIV,HCV,andHBV:thecobasTaqScreenMPX(PCR)testandtheTigrisSystem.Weused transcription-mediatedamplificationtoreviewandcomparethedevelopmentandefficiencyofthese technologies.
ß2014TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(
http://creativecommons.org/licenses/by-nc-nd/3.0/).
* Correspondingauthor.Tel.:+5511993647519;fax:+551150556588. E-mailaddress:gui.albertoni@uol.com.br(G.Albertoni).
ContentslistsavailableatScienceDirect
International
Journal
of
Infectious
Diseases
j o urn a l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / i j i d
http://dx.doi.org/10.1016/j.ijid.2014.04.007
2.1. Real-timePCR
ThefactthatPCRreactionscanbemonitoredinrealtimehas revolutionizedtheprocessofDNAandRNAfragment quantifi-cation.Nucleicacidquantificationusingquantitativereal-time PCR (qRT-PCR) is extremely accurate and reproducible. In qRT-PCR,eachround ofamplificationleadstotheemissionof a fluorescent signal, and the number of signals per cycle is proportionaltotheamountoftargetnucleicacidinthestarting sample.14Thisallowsforaccurateandreproducible quantifica-tionbasedonfluorescence.Themostcommonlyusedfluorescent compoundsinqRT-PCRaretheSYBRGreendyeandtheTaqMan probe.14 qRT-PCR requires an instrumentation platform that consistsofathermalcycler,acomputer,opticsforfluorescence excitation and emission collection, and data acquisition and analysissoftware.14–16
2.2. In-houseNATforHCVdetection
ForthedetectionandquantificationofHCVRNA,17wedesigned anddevelopedaone-stepNATTaqManqRT-PCRmethod.
Using international standards(ACCURUNHCV RNAPositive Control;SeraCare)containing3105IU/mlofHCVRNA(1U=1–3 copies),14 our NAT in-house qRT-PCR TaqMan HCV assay displayedlinearityoverarangeof3.1102to3.1105IU/ml (2.5–5.5log).Thelowerlimitofdetectionofthemethodwas310 IU/ml.InapreviousreportbyWendeletal.,18themethodusedto screen blood donors by NAT testing for HCV RNA displayed linearityovera rangeof5102to5104.Thelowerlimitof detectionofthemethodwas500IU/ml.Inanotherstudy,Paryan etal.19designedanddeveloped anin-house multiplexRT-PCR assayforthedetectionofHCVinplasmasamples.Thatmultiplex assay waslinear between 102 and105 copies/ml (sixof eight
samplesweredetectable), andhad an analytical sensitivityof 200copies/ml.
2.3. In-houseNATforHIVdetection
Wedesignedanddevelopedaone-stepTaqManNATqRT-PCR methodforthedetectionofHIVRNA.20
WerecentlydevelopedaNATTaqManqRT-PCRHIVRNAassay usingasetofinternationalstandards(AcroMetrixHIV-1PanelIU/ mlRNAPositiveControl;AppliedBiosystems)containing1102 to1 107 IU/ml of HIV RNA. Eachpanel member(except the negativecontrol)containsHIV-1atapredeterminedconcentration calibratedagainsttheWHOinternationalstandardforHIV-1RNA. Consequently,theassigned values arereported in international unitspermilliliter(IU/ml).Somestudieshavereportedresultsin unitsthat differ fromtheinternationalunits asdefined by the WHO.21For our in-house NAT TaqMan qRT-PCRHIV assay,we observedlinearityoverarangeof1102to1107IU/mlanda lowerlimitofdetectionof100IU/ml(Figure1).Similarly,Paryan etal.,19usingmultiplexRT-PCR,showedlinearityoverarangeof1 102to1105copies/mland ananalyticalsensitivityof 100 copies/mlforHIVRNA.InexperimentsbyDeCrignisetal.,22aSYBR GreenIMultiplexRT-PCRwassuccessfullyusedtodetectHIVRNA, andwasfoundtohaveananalyticalsensitivityof400copies/mlfor HIVRNA.
2.4. In-houseNATforHBVdetection
WedevelopedaNATin-houseqRT-PCRTaqManHBVDNAassay usinginternationalstandards(AcroMetrixHBVPanelIU/mlDNA PositiveControl;AppliedBiosystems)containing2102to2107 IU/mlofHBVDNA.OurHBVassaydisplayedlinearityoverarange of2102to2107IU/ml,andthelowerlimitofdetectionofthe
methodwas200IU/ml(Figure2).dosSantosetal.23developeda cost-effectiveRT-PCRtesttodetectawiderangeofHBVDNAinthe westernmazonasregion.Theirmethoddisplayedlinearityovera rangeof2103to2109copies/mlandalowerlimitofdetection of2000IU/ml.Yalamanchilietal.24designedarapid,reliable,and sensitiveassayforthedetectionofHBVbyRT-PCR.Thecalibration curvewaslinearoverarangeof1102to1108copies/ml,with anR2valueof0.999.Thelowerlimitofdetectionofthemethodwas 100copies/ml.
3. Currentapproaches
SeveralUSFoodandDrugAdministration(FDA)-licensedNAT assaysarecurrentlyavailableforthescreeningofblooddonorsfor HIV, HCV, HBV, and West Nile virus (WNV). The continued developmentofhighlysensitivescreeningNATsystems,however, ischallenging.25TheFDAhaslicensedseveraltriplex(HIV/HCV/ HBV)automatedNATsystemsforblooddonorscreening.26These includethePCR-basedcobasTaqScreenMPXassayusingthecobas s 201 instrument (Roche Diagnostics GmbH, Mannheim, Germany),andtheProcleixUltrioassay,usingtheProcleixTigris automatedinstrument(NovartisDiagnostics,Emeryville,CA,USA/ Gen-Probe, San Diego, CA, USA) and employing transcription-mediatedamplification(TMA).27–30
3.1. PCR-basedcobasTaqScreenMPXtest
ThecobasTaqScreenMPXtestisamultiplex,multi-dyenucleic acidamplificationtechnology.Samplenucleicacidsandarmored RNA internal control (IC) molecules, which serve as specimen preparation and amplification/detection process controls, are processed simultaneously. A protease solution digests proteins Figure1.StandardcurveoftheHIVRNAconcentration(IU/ml)(slope: 3.20;R2: 0.99).
topromotelysis,inactivatenucleases,andfacilitatethereleaseof RNAandDNAfromviralparticles.Thereleasednucleicacidsbind tothesilicasurfaceofmagneticglassparticles,whichareaddedto thereactionmixture.Thebindingofnucleicacidstotheparticlesis mainlyduetothenetpositivechargeontheglassparticlesurface andnetnegativechargeofthenucleicacidsatthechaotropicsalt concentration and ionic strength of the lysis reaction. A wash reagent removesunbound substances and impurities. Using an elutionbuffer, purifiednucleicacidsarethenreleasedfromthe magneticglassparticlesathightemperatures.31
Afterisolationofthepurifiednucleicacidsfromhumanplasma, cobas TaqScreen Master Mix (MPX MMX) is used for the amplificationand detection of HIV-1(groups Mand O), HIV-2, andHCVRNA,aswellasHBVDNAandICRNA.OncetheMPXMMX is activated by the addition of manganese acetate, reverse transcription (for RNA targets) proceeds, followed by PCR amplification of highly conserved regions of HIV-1 (groups M andO),HIV-2,andHCVRNA,aswellasHBVDNAandICRNAusing specificprimers.31
Simultaneous detection of the amplified nucleic acids is accomplishedby the generationof fluorescentsignals from50 -nucleolyticdegradationofHIV-1-(groupsMandO),HIV-2-,HCV-, HBV-andIC-specificprobes.Twofluorescentdyesareused,one labeling the IC probe and a second labeling all target-specific probes, allowing for the combined identification of each viral target as well as independent identification of the IC. Reverse transcription and amplification reactions are performed with a thermostable recombinant DNA polymerase enzyme. In the presence of manganese (Mn2+), DNA polymerase has reverse
transcriptase and DNA polymerase activities. This allows both reversetranscriptionandPCRamplificationtooccurinthesame reactionmixture.31
Selectiveamplificationoftargetnucleicacidfromthespecimen is achieved by the use of a uracil-N-glycosylase enzyme and deoxyuridinetriphosphate.Theenzymerecognizesandcatalyzes thedestructionofDNAstrandscontainingdeoxyuridine,32butnot DNA containing deoxythymidine or RNA containing ribouri-dine.33,34
DuringPCRamplification,theintermittenthightemperatures duringthermalcyclingdenaturetargetandICampliconstoform single-strandedDNA.Thespecificoligonucleotideprobes,which facilitatesignaldetection,hybridizetothesingle-strandedformof theamplifiedDNA,andamplification,hybridization,anddetection occur simultaneously.33,35,36 During PCR amplification, probes hybridize to specific single-stranded DNA sequences and are cleavedbythe50 to30 nucleaseactivityoftheDNApolymerase whileamplificationisoccurring.Oncethereporterandquencher dyesareseparatedbythiscleavage,thefluorescentactivityofthe reporter dye is unmasked. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signalofthereporter dyeisconcomitantlyincreased.35,36 Real-timedetectionofPCRproductsisaccomplishedbymeasuringthe fluorescence of released reporter dyes representing the viral targets.35,36
3.2. PublishedstudiesthathaveusedthecobasPCRNATsystem
Inarecentstudy,Mu¨lleretal.26usedacommercial,multi-dye nucleicacidamplificationtechnologytestforHBV/HCVandHIV-1/ 2 to assess the analytical sensitivity of the test in samples containinglowconcentrationsofeachvirus.Thestudydescribes theevaluationoftheMPXv2testbytwobloodbanksinEurope: theGermanRedCrossBloodDonorService(Frankfurt,Germany) andtheCentrodeHemoterapiayHemodonacı´ondeCastillayLeo´n (Valladolid,Spain).TheanalyticalsensitivitiesoftheMPXv2test determinedbytheGermanandSpanishbloodbankswere1.1and
3.5IU/mlforHBV,3.9and17.6IU/mlforHCV,and43.3and50.6IU/ mlforHIV-1,respectively.26
Inanotherstudy,Roche37reportedontheanalytical
sensitivi-tiesoftwoversionsoftheMPXtest(MPXv1andMPXv2)inthe detection of HBV, HCV, and HIV-1. In the case of the current commercialNATtest–theMPXv1test–areactivesamplehasto betestedfurtherwiththreeindividual,virus-specificNATtestsin order to identify the viral contamination. One of the major disadvantagesofthisstrategyisthataconfirmedreactivesampleis often non-reactive when tested with discriminatory tests.38–40 Thisdiscrepancyismostlikelytooccurwithsamplescontaining low viralloads,typically samplesfromdonors withoccultHBV infection.39TheMPXv2testthatwasevaluatedinthisstudywas thefirstcommercialmultiplextestabletosimultaneouslydetect andidentifyviralcontaminantsinplasmasamples.TheMPXv2 testthuseliminatesthedrawbacksofthepreviousversionofthe test.26Thelowerlimitsofdetection(IU/ml)oftheMPXv1testwere 3.8,11.0,and49.0forHBV,HCV,andHIV-1,respectively,andinthe caseoftheMPXv2test,thelimitswere2.3,6.8,and46.2forHBV, HCV,andHIV-1,respectively.37,41Previousstudies42,43foundthe MPX v1 test to be more sensitive for HBV than for HCV, and althoughitappearsthattheanalyticalsensitivityoftheUltriotest ismoresensitivethantheMPXtestsforHIV-1,thetwostudiesthat have directly compared themethods43,44reported these differ-encesbetweentheUltrioandMPXv1testsnottobesignificant. Similarly,theanalyticalsensitivitiesoftheGermanRedCrosskits for HBV and HIV-1 detection appear to be significantly more sensitive than those of the MPX v2 test;however, the limited clinicalresearchcomparingthetwotestsshowednodifferencein clinicalperformance.26
TheperformanceoftheMPXv2testforroutinedonorsamples wascomparedwiththetestsonrecordatthetwobloodbanks.26In thelattercase,anHBV-positivesamplewasnotdetectedwiththe testonrecord,whichwastheMPXv1test,althoughtheanalytical sensitivitiesoftheMPXv1andMPXv2testsareverysimilar(95% limit of detection of 3.8 and 2.3 IU/ml, respectively).26 One explanation forthis could bethat the donorin this casewas a chronic HBV carrier with a low HBV titer that was detected intermittently.39Theperformanceofthe MPXv2testfortesting routinedonorsampleswascomparabletobothofthein-housetests oftheGermanRedCrossBlooddonorServiceandtheMPXv1testat CentrodeHemoterapiayHemodonacı´ondeCastillayLeo´n.26
Stramer et al.30 provide a summary of HBV DNA analytical sensitivitiesofcommerciallyavailabletriplexNATassays.These dataarelimitedtothosestudiesusingwell-characterizedWHO stockpreparations.HBVDNAanalyticalsensitivityata95%limitof detectionisapproximately4IU/ml(range3–8IU/ml),andHVCand HIV analyticalsensitivities(95%limitofdetection) are approxi-mately7–22IU/mand42–58IU/ml,respectively.
3.3. ProcleixUltrioassay:TMA
Thestate-of-the-artTMAtechnologyusedintheProcleixUltrio assaywasdevelopedbyGen-Probe,Inc.(SanDiego,CA,USA),our partnerforNATinnovation.TheassaymakesuseoftheProcleix TigrisSystem,whichisafullyautomatedsystemfornucleicacid amplificationanddetectionbasedonTMAtechnology.
targetnucleic acid,and RNA polymeraseinitiatestranscription, synthesizingRNAfromthecDNA.Someofthenewlysynthesized RNAamplificationproductsreentertheTMAprocessandserveas templatesfornewroundsofamplification.Billionsofcopiescan potentiallybegeneratedinlessthananhour.43Detectionfollows on from the amplification step. Acridinium ester (AE)-labeled probesspecificallyhybridizetoamplificationproducts.Different AEvariants are used tolabelIC- and viral-specificprobes.The hybridization protection assay(HPA) process selectively inacti-vates the AE labels on unhybridized probes to minimize backgroundsignal,anddualkineticassay(DKA)technologyallows for the simultaneous detection of both IC-encoded and viral-encodedRNAbythedetectionofabriefflashoflightanda longer-lastingglow,respectively,producedbytheRNA.43
3.4. PublishedliteratureonTMA-basedNATs
A new version of the TMA assay, Ultrio Plus, was recently launchedintheEuropeanUnion(EU),andwewereinterestedto knowwhetherthisnewversionoftheassayhasimprovedsensitivity indetectinglowviralloadHBVcomparedwiththeoriginalassay.In comparisontotheoriginalUltrioassay,theUltrioPlusassayincludes anadditionalreagentthatcontainsconcentratedlithiumhydroxide, whichenhancesthedisruptionofHBVparticlesandthesubsequent releaseofDNAforthecaptureprobetotarget.
Grabarczyketal.4reportedonadirectcomparisonofthetwo TMAassaysforthedetectionofHBV,HCV,andHIVinblooddonors. TheanalyticalsensitivitiesoftheUltrioandUltrioPlusassayswere analyzedusing dilution panels ofWHO internationalstandards withthefollowingconcentrations:WHOHBVgenotypeA:50,15, 5,1.5,0.5,0.15,and0.05IU/ml;WHOHCVsubtype1:100,30,10,3, 1,0.3,and0.1IU/ml;andWHOHIV-1subtypeB:600,200,60,20,6, 2, and 0.6 IU/ml. A comparison of the 50% and 95% limits of detectionbyprobitanalysisinparallellinemodeshowedthetwo versionsoftheTMAassaystohaveequalanalyticalsensitivityto theWHOHCVandHIV-1standards,butinthecaseofHBV,the UltrioPlusassaywasfoundtohavea2.4-foldhighersensitivity thantheUltrioassay.4Theassessmentoftheanalytical sensitivi-tiesofthetwoassaysshowedthe95%limitsofdetectionforHBV DNA,HCVRNA,andHIV-1RNAtobe11.1,9.0,and14.2IU/ml, respectively,for theUltrio assay, and 4.6, 9.3, and 18.5 IU/ml, respectively,fortheUltrioPlusassay.4
Strameretal.42carriedoutacomparativestudyoftriplexNAT assaysintheUSA.Theresultsofthestudyshowedthe95%limitof detectionofHBVDNAtobe10–15IU/ml(95%range,8–40IU/ml)in theUltrioassayand 2–4IU/ml(95%range,2–10IU/ml)for the UltrioPlusassay.TheUltrioPlusassaywasthusmoresensitivefor HBVDNA detectionthan theUltrio assay.According tostudies reported on in theproduct packageinserts, theHCV analytical sensitivitiesforthetwoassaysareequal(2–6IU/ml),asarethe HIV-1analyticalsensitivities(16–40IU/ml).
InacomparativestudyevaluatingtriplexNATsforthedetection ofHBVDNA,HCVRNA,andHIV-1RNAbyXiaoetal.,43theProcleix TigrisSystemoftendetectedHBVatvirallevels below50IU/ml. WhereastheHBVviralloadsinthechronicdonationswerecloseto the NATcut-off (approximately15 IU/ml for the ProcleixUltrio assay).44
4. Discussion
Fullyautomatedtriplex(HIV,HCV,andHBV)NATassayshave beenavailableintheUSAsince2007,andguidelinesregardingthe requirementsforHIV-1andHCVNATassayswereissuedin2010.45 InNovember2012,theFDAissuedguidelinesregardingHBVDNA detectionNATassays,stipulatingthatsuchassaysshouldhavea minimumsensitivityof100IU/ml(approximately500copies/ml).46
Themain advantageof theMPX v2test over otherlicensed blood screening NATs currently being used, is its ability to simultaneouslydetectandidentifytheviraltargetinasample.The need for supplementary viral target discriminatory tests is eliminatedin this assay,asare theassociatedtraining require-ments.Multiplex,multi-dyetestsalsoreducetherequiredsample volumes, which is important in an environment in which the number of viralagents being testedfor by NATshas gradually increasedoverthelastfewyears.26TheMPXv2testiscurrently limitedtofourchannelsanddyes,oneofwhichisusedfortheIC. Consequently,allHIVviruses(HIV-1groupsMandOandHIV-2) are detected in a single channel, and thus further testing is required.26TheRocheMPXtestcarriedoutonthes201platform represents an improved and fully automated NAT blood and plasma screening system.26 As shown in our results, this technology proved to be highly sensitive compared with the technologiesdevelopedin-houseinourgroup.17,20
TheanalyticalsensitivitystudiesontheUltrioandUltrioPlus assay systems showed that the two TMA assays are equally sensitive for the detection of HCV and HIV-1. However, the inclusionofanalkalineshocksteptothevirusparticleduringthe targetcapture processhassignificantly improvedtheanalytical sensitivityoftheassaytoHBVintheUltrioPlusassay.TheNAT efficiencyinbothTMAassayversionsappearstobeoptimalfor HCVandHIV-1.ThedetectionefficiencyoftheoriginalUltrioassay forHBVDNA,however,waspoor,andrangedfrom2%to43%.This range wasincreasedto18% to81%byimproved targetcapture chemistryintheUltrioPlusassay.4Oneexplanationforthisisthat theimprovementofferedbythealkalineshockdependslargelyon thelengthofthedouble-stranded(ds) DNAportion oftheHBV genome.4ThisdsDNAportionvariesevenwithinonecarrier,47and may be even more variable between individuals infected with different HBV variants. Itis thus possible that denaturation of dsDNA is an additional contributing factor to the enhanced sensitivity of the Ultrio Plus assay, and that the remaining variability in limits of detection in the Ultrio Plus assay is a reflection of incomplete denaturation of dsDNA during target capture.Anotherexplanationforthehigherdetectionefficiencyof the Ultrio Plus assay for HBV is the denaturation of a tightly attachedproteintotheHBVgenome47thatotherwisecouldinhibit targetcapture by steric hindrance.Suchan inhibitoryeffect by interferingproteinsdoesnotplayaroleincapturingHIVRNAand HCVRNA,whichmayexplainwhytheNATefficienciesforthese virusesarefoundtobehigherthanthatforHBV.Regardlessofthe explanation,thealkalineshockstephascertainlycontributedtoan enhancedsensitivityintheUltrioPlusassay.
TheresultsofacomparativestudyoftheUltrioandUltrioPlus assays showed that there was no difference in the analytical sensitivitiesofthetwoassaystoHCVandHIV-1,butthattheUltrio Plusassayhadathree-foldhighersensitivitytoHBVthantheUltrio assay.
5. Conclusions
Although the introduction of real-time PCR has led to considerable progress in automating the amplification and detection steps of NATs, nucleic acid isolation remains very labor-intensive when performed manually. Traditional phenol– chloroform extraction and ethanol precipitation methods are complicated, time-consuming, hazardous, and unsuitable for processinghighnumbersofsamples.
amplificationconditions;(4)areproduciblequantitative amplifi-cationassayisverydifficulttoconstructanditremainslaborious andcumbersome.Thus,theautomationofnucleicacidextraction andamplificationtechnologybecomesnecessary.
Acknowledgements
ThisworkwassupportedbygrantsfromFundac¸a˜odeAmparoa PesquisadoEstadodeSa˜oPaulo(FAPESP),ConselhoNacionalde DesenvolvimentoCientı´ficoeTecnolo´gico(CNPq),Coordenac¸a˜ode Aperfeic¸oamentodePessoaldeNı´velsuperior(CAPES),Fundac¸a˜o OswaldoRamos(FOR),andFundodeAuxı´lioaosDocenteseAlunos (FADA).Thefundershadnoroleinthestudydesign,datacollection andanalysis,decisiontopublish,orpreparationofthemanuscript.
Conflictofinterest:Theauthorshavedeclaredthatnocompeting interestsexist.
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