Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Improvement

of

Mycobacterium

tuberculosis

detection

in

sputum

using

DNA

extracted

by

sonication

Graziele

Lima

Bello

_,

_Franciele

_Costa

_Leite

_Morais

_,

_Jonas

_Michel

_Wolf

_,

_Mirela

_Gehlen

_,

Tainá

dos

Santos

Soares

_,

_Maria

_Laura

_Halon

_,

_Regina

_Bones

_Barcellos

_,

Maria

Lucia

Rosa

Rossetti

a_{UniversidadeLuteranadoBrasil(ULBRA),ProgramadePós-Graduac¸ãoemBiologiaCelulareMolecularAplicadaàSaúde,Canoas,RS,}

Brasil

b_{UniversidadeFederaldoRioGrandedoSul(UFRGS),ProgramadePós-Graduac¸ãoemPneumologia,PortoAlegre,RS,Brasil} c_{UniversidadeLuteranadoBrasil(ULBRA),CursodeBiomedicina,Canoas,RS,Brasil}

d_{SecretariadoEstadodoRioGrandedoSul(SES/RS),CentrodeDesenvolvimentoCientíficoeTecnológico(CDCT),PortoAlegre,RS,Brasil} e_{UniversidadeFederaldoRiodeJaneiro(UFRJ),ProgramadePós-Graduac¸ãoemClínicaMédica,RiodeJaneiro,RJ,Brasil}

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Received10May2020 Accepted16August2020

Availableonline12September2020

Keywords: Tuberculosis Sputum DNAextraction Sonication

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Tuberculosis(TB)isoneoftheinfectiousdiseaseswithhighmortalityintheworld.DNA amplificationtechniqueshavebeenusedtoovercomebarrierstothediagnosisofthis dis-ease.However,thesuccessofthesemethodologiesishighlydependentontheDNAobtained fromthesample.ThisstudywascarriedouttoverifywhethertheDNAextractedby soni-cation(inhousemethod)couldyieldsuitableDNAforamplificationbyreal-timePCR(qPCR). SixtysputumsamplesweresubmittedtoDNAextractionusingsonicationcomparedtoa commercialmethod(Detect-TBkit,Labtest/MG-Brazil).AllDNAsampleswereamplifiedby qPCRforIS6110region(IS6110-qPCR/SYBRGreenassay).Outof60samples,40werepositive forTB;ofthese,allhadpositiveresultswhenextractedbysonication(100%)and80%when extractedbythecommercialmethod.Thelimitofdetection(LOD)ofMycobacterium tuber-culosis(H37Rvstrain)byqPCRwas14CFU/mLwhentheDNAwasextractedbysonication, comparedtocountlesscolonieswhenextractedbycommercialkit.Inconclusion,the soni-cationprotocol(withoutpurificationstep)provedtobeasimple,fast,andsuitablemethod forobtainingDNAforuseinqPCRfromsputumsamples.

©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddresses:[email protected](T.S.Soares),[email protected](M.L.Rossetti).

https://doi.org/10.1016/j.bjid.2020.08.006

1413-8670/©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background

Tuberculosis(TB)isoneofthemaincausesofdeathandthe leadingcausefromasingleinfectiousagentworldwide.1 Mil-lionsofpeoplecontinuetofallsickwithTBeachyear,2_more than1.7billionpeople(about25percentoftheworld popula-tion)areestimatedtobeinfectedwithM.tuberculosis.In2017, 10millionindividualsbecameillwithTBand1.6milliondied.3 Early diagnosis is one of the strategies to combat the disease.4Methodologiesbasedontheamplificationofnucleic acidsbyPolymeraseChainReaction(PCR)forthedetection ofM.tuberculosisDNAhaveimprovedthediagnosisandcan overcomeproblemsassociatedwithclassicalmethods.5_The useofmoleculartechniques,besidescontributingtodetect theincreaseinthenumberofTBcases,alsoallowstoquickly characterizeresistanceanddefinetheexistinggenotypesina givencommunity.6

However,differences in sensitivityand specificity when usingPCRtechniquesareareality.Therearemanyreasonsfor thisvariability.OneistheappliedDNAextractionprotocol.7 TheDNAextractionmethodmustprovideagoodyieldand DNAofquality,eliminationofPCRinhibitors,implementable asalaboratorytechnique,resultinginageneticmaterialof good quality for additional molecular analyses, whilestill beingsimpleandfast.8_{Manycommercialmethodsare} avail-able(QIAGENQIAmpDNAminikit,NucleicAcidandProtein Purification-Macherey,Nagel,DynaMagTM-2Magnet, Detect-TBkit),whereasDNApurificationwithsilica,magneticbeads, Chelexbeads,amongothers,havebeendescribedtoimprove DNAquality.7,9,10_{Also,manylaboratoriesusein-house} meth-odsforthisstep,mainlytocurbcosts.11–13

The DNA extraction method that uses the cellular disruption by sonication has also been used in several laboratories,14–16_{butthereisstilldoubtabouttheefficacyof} DNAextracteddirectlyfromsputumclinicalsamples(which haveawidevarietyofmicroorganisms,manyimpurities,and dependenceongood collection)foruseinPCR,asthe pro-tocoldoesnotincludeapurificationstep.17,18_{Therefore,this} studyaimedtocompareasonicationtechniqueasthemethod forobtainingDNAfromsputumsampleswiththecommercial method(silicaresin-Detect-TBkit,marketedbyLabtest/ MG-Brazil).IS6110–qPCR/SYBRGreenwasusedasaconfirmatory methodfordetectingbacterialDNA.19

Materials

and

methods

Sixty sputum samples were collected from patients who attendedtheDepartmentofTisiologyandLeprosyinCanoas, SouthernBrazil.Initially,toevaluateDNAextractionwiththe twoprotocolsfromthesamebacterialconcentration,20 spu-tum samplesknown tobe from TB-negativepatients were contaminatedwith25␮L(100CFU/mL)ofanaqueous suspen-sionofMycobacteriumbovisstrain.

qPCRamplificationwasverifiedin20sputumsamplesfrom patientswithtuberculosis.Todeterminethelimitofdetection,

20negativesampleswerecontaminatedwith25␮Lof10-fold dilutionsofM.tuberculosisreferencestrainH37Rv.

Thesputumsamplesusedinthisstudywerealsoassessed on smearslides,culture,and GeneXpertMTB/RIF (Cepheid, EUA).TheflowchartoftheanalysisispresentedinFig.1.

Samplepreparation

Thesputumsamplesweredecontaminatedandtreatedwith 4%sodium hydroxide (NaOH)and PBS (Phosphate-buffered saline)solutioncontainingsodiumchloride,potassium chlo-ride, sodium phosphate, monobasic potassium phosphate, andultrapurewater,atanadjustedpHof7.4.Briefly,atotal of500␮Lofsputumwas transferredtoa1.5mLmicrotube and added the corresponding volume of NaOH 4% solu-tion,followedbyvortexhomogenization,andincubationat 37◦Cfor15min.Subsequently,itwascentrifuged3000gfor 15minand the supernatant was discarded. Thepellet was resuspendedin500␮LofPBSbuffer andhomogenizedona vortex.

DNAextraction

ProtocolA-silicaresin(Detect-TBkit,Labtest/MG-Brazil)

The silica resin method was described by Michelon et al. (2011)20_{asanadaptationoftheBoometal.protocol(1990).}21 Briefly,theprotocolconsistedofacelllysisstepwitha solu-tion containing8Mguanidinehydrochloride,pH6.4,40mM EDTAand2g/dLpolyoxyethyleneoctylphenylether,followed byincubationinathermoblockat100◦Cfor10min,aiming atthereleaseandsolubilizationofthegenomicDNA. Purifi-cationofthereleasedDNAwascarriedoutthroughspecific uptakebyasilicaresininthepresenceofchaotropicagents. Subsequently,awashsequencewasperformedwithdistinct solutions: the first containingguanidinehydrochloride, the secondcontainingbufferedsodiumchlorideconcentrateatpH 7.0andpolyoxyethylenesorbitanmonolaurate0.1%,andthe thirdcontainingconcentratedsodiumchlorideandbufferedat pH7.5withpolyoxyethylene0.1%monolaurate.Afterwashing, thepurifiedDNAwaselutedin25␮Lofultrapurewaterand frozenat−20◦Cuntiluse.

ProtocolB-sonication(in-housemethod)

Amicrotubecontaining500␮Lofsputumsamplepretreated asmentionedabove,wasplacedat95◦Cfor20min.Afterthis step,itwasplacedinthesonicationbathfor15min,andthen itwascentrifugedfor5minat3000g.Subsequently,100␮Lof thesupernatant(containingtheDNA)wastransferredtoanew microtubeandfrozenat−20◦_Cuntiluse.

QuantitativeassessmentofextractedDNA

The DNA quantification was performed by spectropho-tometry. The DNA absorbance and concentration were evaluatedfornucleicacidpurity(idealratio:A260/A280≥1.8 and A260/A230=2). Samples were quantified in the SpectraMaxPlus® (Eppendorf) spectrophotometer using 2␮LofDNAaccordingtothemanufacturer’sinstructions.

60 sputum samples 20 samples without TB M. bovis contamination 10 samples Protocol A 10 samples Protocol B 20 samples without TB M. tuberculosis H37Rv contamination Ten-fold dilutions: LOD

10 samples ten-fold dilution Protocol A 10 samples ten-fold

dilution Protocol B 20 samples withTB 10 samples Protocol A 10 samples Protocol B

Fig.1–Flowchartofthesamplesusedinthestudy.

ProtocolA:Detect-TBkit,Labtest/MG-Brazil;ProtocolB:Sonication;LOD:limitofdetection.

Limitofdetection(LOD)

Colonies of M. tuberculosis H37Rv strain grown in Ogawa Kudohculturemediumwerescrapedand placedinaglass tubecontainingbeadstosolubilizeandhomogenizebacterial colonies,andaddedultrapurewater(thevolumecanbe stip-ulatedbytheoperator).Theturbidityofthebacterialsolution wascomparedtotheturbidityoftheMacfarlandnumber1 scale(widelyusedinmicrobiology,especiallyforconventional antibiograms).Ten-fold dilutions(turbidityscale1solution) wasperformed.Fromthesedilutions,25␮Lweremixedto20 TB-negativesputumsamples.Thesame volumeof25␮Lof thedilutionswasinoculatedin7H10culturemedium,andthe CFUswasassessedforeachdilution.

Real-timePCR(IS6110–qPCR/SYBRGreen)

Forevaluationoftheviability ofthe extractedDNA,IS6110 real-timePCRwasperformedwiththeStepOneReal-TimePCR Systems(ABAppliedBiosystems)equipmentandtheamplified productsweredetectedusingtheSYBR® Green(fluorophore). Thereactionwasstandardizedinafinalvolumeof20␮L con-taining10␮LofFastSYBR® GreenMasterMix,4pmolofeach primer (INS1 5 ’CGTGAGGGCATGGAGGTGGC 3 and INS2 5 ’GCGTAGGCGTCGGTGACAAA 3) and 5␮Lofextracted DNA andquantified.Theconditionsoftheamplificationwere as follows:activation ofthe enzymeat95◦C for20s, denatu-ration at95◦C for 1s, and finally annealing and extension

at 62◦C for20s. For each run, a negative reaction control (PCRmixcontainingultrapurewater)andapositivecontrol reference(M.tuberculosisH37Rv-80ng/␮L)wereused. Ampli-ficationanddissociationcurveswereanalyzedwithStepOne softwareversion2.3.Fortheanalysisoftheamplificationcurve, the parameters ¨threshold¨(detectablefluorescencelevel)and ¨baseline¨(definedlimitofPCRcycles)wereusedtodetermine theCt(cyclethreshold)ofeachsample.Thedissociationcurve wasusedtoobservethepossibleamplificationofnon-specific productsorprimerdimers. Forstandardizationoftheideal concentrationofprimersat4pmol,itwasusedtheanalysisof thedissociationcurveofknownsamplesandtheanalysisof ampliconsin1.5%agarosegel,stainedwithethidiumbromide.

Standardcurveandefficiency

Thesetupofthestandardcurvewasperformedaccordingto themanufacturer’srecommendations(ABApplied Biosystems-Thermofisher Scientific)—five points and in duplicate—for assessingDNAamplificationprotocol.Ten-folddilutionswere performedfromthequantifiedDNAofM.tuberculosisstrain H37Rv(usingasagenomicmarkertheelementofinsertion IS6110ofthebacterium,byqPCR).ThePCRmixturecontained 5␮LofDNAfromeach dilutionforeachreaction,and15␮L ofthe mix, totaling thefinal volumeof20␮Lper reaction. Efficiency, calculatedusingStepOne softwareversion2.3,was of96.15%(referencevalue:90–100%),R2_{of0.97(indicatesthe}

stan-Table1–ComparisonbetweentheaveragesofthedegreesofpurityandDNAconcentrationsobtainedbythetwo protocolsappliedtoclinicalsamplescontaminatedwithM.bovisstrain.

Protocols A260/280(≥1.8) A260/230(<2.0) DNAng/␮L Intensityamplicons

electrophoresisgel

A 1.67±0.35 0.14±0.12 58.83±76.65 ++

B 0.97±0.07 0.74±0.33 340.43±154.34 +++

p-values <0.01* <0.01* <0.01*

Protocols:A)Detect-TBKit;B)Sonication.

∗ _{Significantstatisticaldifference.}

dardcurve andthe dataofindividual Ctpoints),−3.418 of slope(indicatestheefficiencyofamplificationbyPCRassay) andY-interof16.87(indicatestheexpectedCtforthesample).

Ethicalaspects

ThestudywasapprovedbytheLutheranUniversityofBrazil (ULBRA)researchethicscommittee(approvalnumber 2011-340H).

Statisticalanalysis

AlldatawereanalyzedusingIBM® SPSS® statistics(version 21.0).TheShapiro-Wilktestwasusedfortheanalysisoftype ofdatadistribution.Student’sT-testforindependentsamples andMann-Whitneytestwereusedtoidentifypossible differ-encesofDNAconcentrationsandCtsobtainedbetweenthe twoprotocolsevaluated.Alltestsperformedwereinterpreted usingatwo-sidedsignificancelevelof5%(p<0.05).

Results

TheqPCR amplificationof the DNA extracted from the 20 contaminatedsampleswithM.bovis(10DNAsextractedper protocol) showed that DNA extracted by Protocol A (silica resin) wasdetected in8 ofthe 10 samples(80.0%) and all 10DNAsextractedbyProtocolB(sonication)wereamplified (100%).

ThespectrophotometricquantificationoftheDNAs ampli-fiedandpurityevaluationbythedifferentprotocolsareshown in(Table1).RegardingDNApurityatwavelength260/280nm, significantdifferenceswerefoundbetweentheresultsoftwo protocols (1.67±0.35 vs. 0.97±0.07; p<0.01). At the wave-lengthof260/230 nm, a statisticaldifference was detected betweenthedataevaluated,ProtocolAdifferedfromProtocol B(0.14±0.12vs.0.74±0.33;p<0.01).Therewasasignificant differenceinDNAconcentrationbetweenthetwoprotocols usedintheanalysis(58.83ng/␮L±76.65ng/␮Lvs.340.43ng/␮L ±154.34ng/␮L;p<0.01).

TheamplificationsCtsbyreal-timePCRandthemeansof eachDNA extractionprotocol are shownin(Table2). Com-paringCtsofprotocol1vs.2,therewasstatisticaldifference (27.73±2.48vs.26.92±0.89;p<0.01).

RegardingtheLOD(usingten-folddilutionsoftheM. tuber-culosisstrainH37Rv),withprotocolBtheqPCRassaydetected upto14CFUs(1:1000dilution),whereaswithProtocolAthe LODwascountlesscolonies.

Table2–CTscorrespondingtotheextractionprotocols appliedfromDNAextractedfromclinicalsamples contaminatedwithM.bovisstrain.

ProtocolA ProtocolB

Detect-TBKit Sonication

26.48 27.25 26.21 27.16 24.87 27.10 – 27.27 31.49 27.21 25.27 27.23 27.58 27.25 30.56 27.28 – 24.40 29.42 27.14 27.73±2.48 26.92±0.89p-value=0.01*

∗ _{Significantstatisticaldifference.}

Amongthe20samplesevaluatedfrompatientswithTB, DNAextractedwithProtocolAwasdetectedTBinsevenof the10(70.0%)samplesandinall10(100%)DNAextractedwith ProtocolB(Table3).

DNApurityandquantificationextractedfromM. tubercu-losissputumsamplesfollowedthesamestandardsasthose ofM.boviscontaminatedsamples.Table4showsthemean purityandquantificationofthenucleicacidextractedwith the twoprotocols.Regarding DNApurityatthewavelength of260/280nm,therewassignificantdifferencebetweenthe twoprotocols(2.19±1.62vs.1.09±0.21;p<0.01).When ana-lyzing DNApurityatwavelength260/230 nm,therewasno significantdifferencebetweenthetwoprotocols(0.07±0.04

vs.0.68±0.50;p<0.01).ConcerningDNAquantification,there was a significant difference (20.75 ng/␮L± 11.74 ng/␮L vs.

37.01ng/␮L±36.91ng/␮L;p<0.01)betweentheprotocolsused intheanalysis.

TheresultsoftheCtsfortheDNAextractedsamplesfrom patientswithTBusingthetwoprotocolsareshowninTable5. The difference observedbetween ProtocolA vs. Bwas not significant(29.03±4.14vs.26.48±4.44;p=0.23)when consid-eringthesampleswithMycobacteriumDNAdetected.

Discussion

We assessedin this study if extraction DNA from sputum samples (simple and low-cost technique) with sonication wouldprovideDNAsuitableforthedetectionoftheM. tuber-culosis by qPCR,through the identification of the insertion

Table3–Real-timePCRdetectionofM.tuberculosisDNA insamplesfrompatientswithTB:resultsafterDNA extractionbytwoprotocols.

Clinicalsputum sample

ProtocolA ProtocolB

Detect-TBKit Sonication

1 + + 2 + + 3 + + 4 + + 5 – + 6 + + 7 – + 8 + + 9 + + 10 – +

sequence IS6110, being reported as the most specific and widely explored genetic marker (number of IS6110 copies pergenomemightinfluencethesensitivityofthetest).22,23 Sputumisthemostcommonbiologicalsampleusedforthe diagnosisofpulmonaryTBdisease.24 _{Ascomparedtourine} andblood,sputumisthickerandmoreviscousbutprovides betterinsightintoTBandotherrespiratory-relateddiseases.25 WhenuseddirectlyforDNAextractionandsubsequent detec-tionbyPCR,itisknownthatsputumsamples,presentbarriers related to the presence of internal Taq inhibitors, as well aspresenceofseveralmicroorganisms thatare partofthe microbiotaoftherespiratoryandoraltracts.26–28_Thus,sample preparationandchoiceofanefficientmethodforDNA extrac-tionisoneofthemainchallengesforthemoleculardiagnosis ofTB.29

Regarding the use of the available commercial kits, it is known that they are generally preferred because they providegreaterreproducibility,qualitycontrol,and automa-tionpotential.30 _{Silica matrices(Detect-TBkit,marketed by} Labtest/MG-Brazil-ProtocolA)provideefficientandselective bindingofnucleicacids(NA),havingascharacteristicthe pro-cedureofpurificationofDNA.31_{Themechanisminvolvedin} thistechniqueistheaffinitybetweennegativelychargedNA andpositively chargedsilica material,resultinginselective bindingofNA.32 _{Inclinicalapplications, evenusing kits,to} obtainhighyieldsofNAisstillchallenging,sincesputum sam-plesoftenexhibitalowfinalconcentrationofDNA,whichis maskedagainstalargebackgroundofcontaminants.

In our study, there was variability in some standards regardingthequantitativeevaluationoftheextractedDNA. Nonetheless,ProtocolBprovidedahigheramountofextracted

Table5–CTscorrespondingtotheextractionprotocols appliedfromDNAextractedfromclinicalsamplesfrom patientswithTB.

Sample ProtocolA ProtocolB

Detect-TBKit Sonication

1 33.18 28.43 2 33.80 35.28 3 32.10 29.32 4 28.06 27.56 5 – 24.55 6 26.84 27.06 7 – 20.03 8 26.75 27.97 9 22.49 23.88 10 – 20.77 29.03±4.14 26.48±4.44p-value=0.23

DNA,lowerCtsintheplotamplification(byqPCR)andprofiles offragmentswithamoreuniformaspect, whenvisualized in electrophoresis. In line with our findings, the study by Krinitsinaetal.(2015)33_{showedthatthepurityvalues and} someDNA concentrationsmeasuredbyabsorbance,clearly differfromthevisibledataofreal-timePCRand electrophore-sisdetection(fluorescenceintensityofamplicons).Depending on the extraction methodapplied, it isimportant topoint outthatpurificationofDNAmoleculesisknowntobe influ-enced bytheirspecificitiesofsize,nucleotide composition, topology, and association with proteins.30 _{We believe that} reagentresidueswithdifferentchemicals,ortheneedfor fur-therwashesduringtheapplicationoftheprotocols,mayhave influencedtheabsorbancereading.Therefore,aquantitative evaluationoftheextractedDNAcannotbeevaluatedasan absoluteparameter.

AccordingtothestudybyAldousetal.(2005),34_itwas possi-bletoconcludethatthepurificationstepusingsilicaresinwas notnecessaryfortheextractionofDNAfromsputumsamples beforethePCRtechniqueforidentificationofM.tuberculosis

sinceitmakesthemethodmoreextensiveandimpairs labo-ratoryroutine.ComparingtheuseofKit(Detect-TB–Protocol A)within-housemethod(Sonication–ProtocolB),itwas con-cludedthattheDNAextractedbyKitpresentedhigherpurity, buttheamountofextractedDNAwas,inmanycases, incon-sistentfordetection.32_{InastudybyPsifidietal.(2015),}35₁₁ differentprotocolsweretestedforDNAextractionandfurther usedingenomicand microarraysequencing.Amongthem, theonethatobtainedtheshortestexecutiontime,lowestcost, andthebestresultswerethein-houseprotocol,asourresults alsodemonstrated.

Table4–ComparisonbetweentheaveragesofthedegreesofpurityandtheDNAconcentrationsobtainedbyprotocols appliedtoclinicalsamplesofpatientswithTB.

Protocols A260/280(≥1.8) A260/230(<2.0) DNAng/␮L Intensityamplicons

electrophoresisgel

A 2.19±1.62 0.07±0.04 20.75±11.74 ++

B 1.09±0.21 0.68±0.50 37.01±36.91 +++

p-values <0.01* <0.01* <0.01*

Protocols:A)Detect-TBKit;B)sonication.

The analyses byLiu et al. (2013),36 _{Greenly, and Tester} (2015)37_{showedthattheuseofultrasoundallowscellwallsto} becleavedbyshockwaves,createdbyrapidchangesin pres-surecausedbysonication.ThestudybyGrangeretal.(2004)15 reported that the use of sonication improved mycobacte-rial DNAbyremovingbackground bacteriaand other cells. In addition to these experiments, studies by Pchelintsev, Adams,and Nelson (2016)17 _{showed that the use of} soni-cationminimizes the risk of cross-contamination between samples. Daiet al. (2016)16 _{concludedthat the} sonication-basedtechniqueisconsideredmorepromising,efficient,and economicalforhomogeneouscelldecomposition,inashort period.

Regardingthelimitofdetection(LOD)ofqPCR/SYBRGreen assay,ProtocolBprovidedagooddetectioncapacity(upto14 CFU/mL),evenfromsmalleramountsofDNA(1:1000dilution), consumedfewerreagents,withfewerexecutionsteps,proving tobeeffective,lesslaborious,andfastmethodthatpresented greater potential for application in sputum samples.8 _The LODobtainedinourstudywassimilartothatfoundinthe studybyChakravortyetal.(2017),38_{whichanalyzedthe} per-formanceofXpertMTB/RIFUltraasasuitablepoint-of-care (POC) assay for TB diagnosis, and obtained a LOD of 15.6 CFU/mL.

We recognize potential study limitations. However, our findingsareimportantforguidingthechoiceofDNA extrac-tion methodapplicable toclinical sputumsamples. Future studiesmayconsolidateourfindings,thusallowinggreater understandingoftheneedforNApurificationformolecular detectionofpotentialmicroorganismsthatcauseinfectious diseases.

Conclusions

and

perspectives

Identificationtechniques based on molecular biology have been established as potential tools for diagnosis. In light of this, this study showed that the extraction of DNA by sonication, even without presenting purification steps during the execution of the technique, provided suitable geneticmaterialforidentificationbyreal-timePCR, present-inghigherDNAconcentrations,lowercostsandcomplexity of execution. Thus, it is one of the most promising DNA extraction techniques for use in clinical sputum samples from patients, presentinghigh applicability to the diagno-sis of TB, as well as the possible potential for molecular epidemiologyanalyses,suchasWholeGenomeSequencing (WGS).

Funding

Graziele Bello and Jonas Michel Wolf were supported by CAPES(Coordenac¸ãodeAperfeic¸oamentodePessoaldeNível Superior)/MEC/Brazil-Fellowship.Financingcode001.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorswishtothankthePostgraduateProgramin Cel-lular and Molecular Biology Applied to Health - Lutheran UniversityofBrazil(ULBRA),CenterforScientificand Tech-nological Development (CDCT-RS), and the Department of TisiologyandLeprosyofCanoas,Brazil.

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