Loop-mediated isothermal amplification (LAMP) reaction as ...

Loop-mediated isothermal amplification (LAMP) technology was introduced in the year 2000 with the aim to improve nucleic acid amplification ... Skiptomaincontent Advertisement SearchallBMCarticles Search Loop-mediatedisothermalamplification(LAMP)reactionasviablePCRsubstitutefordiagnosticapplications:acomparativeanalysisstudyofLAMP,conventionalPCR,nestedPCR(nPCR)andreal-timePCR(qPCR)basedonEntamoebahistolyticaDNAderivedfromfaecalsample DownloadPDF DownloadPDF Researcharticle OpenAccess Published:22June2020 Loop-mediatedisothermalamplification(LAMP)reactionasviablePCRsubstitutefordiagnosticapplications:acomparativeanalysisstudyofLAMP,conventionalPCR,nestedPCR(nPCR)andreal-timePCR(qPCR)basedonEntamoebahistolyticaDNAderivedfromfaecalsample PhiawChongFoo  ORCID:orcid.org/0000-0002-5867-98161,2,A.B.NurulNajian2,3,NuraminA.Muhamad4,MarianaAhamad1,MaizanMohamed5,ChanYeanYean2,6&BoonHuatLim7  BMCBiotechnology volume 20,Article number: 34(2020) Citethisarticle 14kAccesses 19Citations Metricsdetails AbstractBackgroundThisstudyreportstheanalyticalsensitivityandspecificityofaLoop-mediatedisothermalamplification(LAMP)andcomparesitsamplificationperformancewithconventionalPCR,nestedPCR(nPCR)andreal-timePCR(qPCR).AlltheassaysdemonstratedinthisstudyweredevelopedbasedonSerine-richEntamoebahistolyticaprotein(SREHP)geneasstudymodel.ResultsAsetofSREHPgenespecificLAMPprimersweredesignedforthespecificdetectionofEntamoebahistolytica.Thissetofprimersrecorded100%specificitywhenitwasevaluatedagainst3medicallyimportantEntamoebaspeciesand75otherpathogenicmicroorganisms.TheseprimerswerelatermodifiedforconventionalPCR,nPCRandqPCRapplications.Besides,3differentpost-LAMPanalysesincludingagarosegelelectrophoresis,nucleicacidlateralflowimmunoassayandcalcein-manganesedyetechniqueswereusedtocomparetheirlimitofdetection(LoD).OneE.histolyticatrophozoitewasrecordedastheLoDforallthe3post-LAMPanalysismethodswhentestedwithE.histolyticaDNAextractedfromspikedstoolsamples.Incontrast,noneofthePCRmethodoutperformedLAMPasbothqPCRandnPCRrecordedLoDof100trophozoiteswhiletheLoDofconventionalPCRwas1000trophozoites.ConclusionsTheanalyticalsensitivitycomparisonamongtheconventionalPCR,nPCR,qPCRandLAMPrevealsthattheLAMPoutperformedtheothersintermsofLoDandamplificationtime.Hence,LAMPisarelevantalternativeDNA-basedamplificationplatformforsensitiveandspecificdetectionofpathogens. BackgroundDNA-baseddetectionmethodhasbeenwidelyusedfordiagnosisofinfectiousdiseasesduetothepresenceofspecificDNAsequencesinpathogensthatcanservedasreliabledetectionbiomarkers[1,2].Thisdetectionmethodisusuallyaccompaniedwithamplificationtechnologysuchaspolymerasechainreaction(PCR),oneofthemostimportantscientificadvancesinmolecularbiology.PCRhasestablisheditsmolecularcompetencyintermofdetectionsensitivityasitcouldamplifyevenasinglegenecopy[3].Despiteitspopularityindiseasediagnostic,PCRamplificationpossessesseveralinherentdrawbackssuchasprimermismatchduetohighDNAsimilarityamongspeciesandlowincopynumberofspecificgeneforpathogenidentification.ThesedrawbackshavelaterpavedthewayfortheemergencesofseveralinnovatedPCRsuchasnestedPCR(nPCR)andreal-timePCR(qPCR).TheinventionofnPCRwastoincreasetheamplificationefficacyintermofdetectionlimit(LoD)andamplificationspecificity.PCRcycleswhichexceed35forproductionoflargerquantityofproductcouldcausegenerationofundesirablesecondaryamplicon[4].nPCRcouldenhancetheamplificationsensitivityandprimingspecificitybyincorporating2successivePCRreactionsusing2primersetsforasinglegenetarget[5].Despiteaddressingseveraladvantages,nPCRwasnotcommonlyusedfordiseasediagnosticsduetoitslongturnaroundtime,anditstwo-step-proceduremadeitsusceptibletoampliconcontamination[6].Real-timePCR(qPCR)techniqueontheotherhandiswellknownwithitsconcurrentdetectionandquantificationofDNA.Itssimultaneousampliconanalysisduringamplificationhassignificantlyshortenedtheturnaroundtimebyobviatingpost-amplificationagarosegelelectrophoresis.InspiteofhavingcomparableperformanceasnPCR[7,8,9],qPCRismachine-dependentwhichisoftenexpensiveandrequiresregularmaintenance[10,11].Loop-mediatedisothermalamplification(LAMP)technologywasintroducedintheyear2000withtheaimtoimprovenucleicacidamplificationefficacyintermofsensitivityandspecificity[12].ThistechnologyhassinceincorporatedintodiagnosticassaydevelopmentfordetectionofmanymedicallyimportantcommunicablediseasessuchasSalmonella Typhimurium[13],pathogenicLeptospira[14],Enterococcusspp.[15],andtoxigenicVibriocholerae[16].IncorporationofLAMPamplificationinreplacingPCRnotonlyeliminatedtheneedsofsophisticatedthermalcycler,itsDNAamplificationefficiencybeyondexponentialhadsignificantlyshortenedtheamplificationduration[12].TheseestablishedLAMP-basedassayswerereportedtobehighlyspecificandsensitive.Besides,thenatureofLAMPthatcouldsynthesisDNAwithauto-cyclingstranddisplacementactivityhaseffectivelyeliminatedtheneedforcostlythermocyclersandtedioustechnicaloptimisationofcyclingconditions[17].TheefficacyofthistechniqueusedfordiseasediagnosticwasfurtherverifiedwhendevelopmentandassessmentofassayusingLAMPperformedbyLiangetal.[18],RiveraandOng[19]andSinghetal.[20]fordetectionofEntamoebahistolyticaalonehavedemonstratedoutstandingcompatibility.AlthoughpreviousstudieshavedemonstratedthebetteramplificationefficiencyofLAMPascomparedtoPCR,thereisyetacomprehensivereporttoevaluatethesensitivitiesamongLAMP,conventionalPCR,nPCRandqPCR.Therefore,theaimofthisstudyistocomparetheanalyticalsensitivityofLAMPassaywith3differentvariantsofPCRandconcurrentlydeterminetheperformanceofLAMPusingagarosegelelectrophoresis,nucleicacidlateralflowimmunoassayandcalcein-manganesedyetechniquesaspost-LAMPanalyses.Toensuretheequityofevaluation,alltheassaysprimersweredesignedtobindonsimilarlocationofSerine-richEntamoebahistolyticaprotein(SREHP)gene.TheperformanceoftheseassayswasevaluatedusingDNAisolatedfromstoolsamplesspikedwithE.histolyticatrophozoites.MethodsReagentsandapparatusGoatanti-mouseIgGsecondaryantibody,streptavidinandfluoresceinisothiocyanate(FITC)IgG1monoclonalantibodyusedfordevelopmentofnucleicacidlateralflowimmunoassayorcommonlynamedaslateralflowdipstick(LFD),werePierceThermoFisherScientific(Massachusetts,USA)products.The40 nmcolloidalgoldsolutionusedwasfromKestrelBioSciences(Thailand),Westernblockingreagent(WBR)wasfromRoche(Indianapolis,USA)andbovineserumalbumin(BSA)waspurchasedfromAmresco(Solon,USA).Betaine,mineraloil,sodiumazide(NaN3),polyvinylalcohol(PVA),polyvinylpyrrolidone(PVP),TritonX-100,Tween-20,sucrose,andothercommonchemicalswerefromSigma(St.Louis,USA).Allthechemicalsandreagentsusedinthisstudywerepreparedusingultrapurewater(>18MΩ)fromaMilliporeMilli-Qwaterpurificationsystem(Billerica,USA).Meanwhile,materialsusedforconstructionofLFDincludingcellulosefiberpads,glassfiberpads,andnitrocellulosemembranecardHF135,werealsoMilliporeproducts.Alllabelledandnon-labelledoligonucleotidesweresynthesisedbyIntegratedDNATechnologies(Singapore).RecombinantTaqDNApolymerase(ThermoFisher,USA)wasusedaspolymeraseenzymeforconventionalPCRandnPCRamplification,andthesereactionswerecarriedoutusingMastercyclernexusgradientthermocycler(Eppendorf,Germany).Meanwhile,qPCRwasperformedusingQuantiFastSYBRGreenPCRKit(Qiagen,Germany)andthereactionwascarriedoutusingCFX96TouchReal-TimePCRDetectionSystem(Bio-Rad,California,USA).TheLAMPBstDNApolymerasewaspurchasedfromNewEnglandBiolabs(Massachusetts,USA)andtheamplificationwereperformedusingCole-Parmerchillingheatingblock(Illinois,USA).TheconventionalPCR,nPCRandLAMPampliconswereanalysedusingagarosegelelectrophoresissystem(OwlSeparationSystems,USA)andvisualisedusingAlphaInnotechChemiImager5500UVilluminatorandimagecapturingunit(California,USA).TheLFDwaslinedwithgoatanti-mouseIgGsecondaryantibodyandstreptavidinmanually,andwerecutintostripsusingMatrix2360programmablestripcutterfromKinematicAutomation(TwainHarte,USA).Calcein-manganesedyeusedforpost-LAMPanalysiswaspreparedusingcombinationofcalceinindicator(Merck,USA)andmanganese(II)chloride(MnCl2)(Merck,USA).EntamoebaspeciesandothermicroorganismstrainsAllthemicroorganismisolatesusedinthisstudyarelistedinTable 1.TheseisolateswerefromtheDepartamentodeMedicinaExperimental,FacultaddeMedicina,UniversidadNacionalAutónomadeMéxico,Mexico;theLondonSchoolofHygieneandTropicalMedicine,London,UK;theDepartmentofMedicalMicrobiologyandParasitology,SchoolofMedicalSciences,UniversitiSainsMalaysia,Malaysia;andtheInstituteforMedicalResearch,Malaysia.E.histolyticaHM-1:IMSSwasusedaspositivecontrolwhilelyophilisedE.disparSAW760andE.moshkovskiiLaredowereusedasnegativecontrolsinthisstudy.DNAofE.histolyticawasisolatedfromaxenicallygrownE.histolyticaHM-1:IMSSwhileDNAofE.disparwasisolatedfromlyophilisedE.disparSAW760usingQiagenQIAampDNAStoolextractionkit(Germany).BothorganismswerereceivedfromtheDepartamentodeMedicinaExperimental,FacultaddeMedicina,UniversidadNacionalAutónomadeMéxico,Mexico.Meanwhile,DNAofE.moshkovskiiLaredowasgivenbytheLondonSchoolofHygieneandTropicalMedicine,London,UK.DNAofothermicroorganismswereisolatedfrompurebacteriacultureusingNucleoSpinTissueDNAExtractionkit(MACHEREY-NAGELGmbH&Co.KG,Germany).TheseDNAswereusedasnegativecontrolsforverificationofconventionalPCR,nPCR,qPCRandLAMPprimersspecificity. Table1ReferenceorganismsusedinthisstudyandanalyticalspecificityevaluationresultsFullsizetablePrimersdesignSREHPgenewasselectedasthetargetgenefordetectionofE.histolyticainthepresentstudyduetoitshighspecificityinbothinsilico(BLASTsearch)andempiricalevaluation(PCRandLAMPamplification)comparedtootherE.histolyticagenes.AlltheprimersusedinthisstudyweredesignedbasedontheconservedregionofthereportedSREHPgene(GenBankaccessionno.M80910.1,M34438.1,XM_643162.2,AB253474.1,AK420158.1,AK420282.1,AK420358.1,AK420741.1).TheprimersetusedforLAMPapplicationwasadoptedfromapreviousstudybyFooetal.[21]andthelocationofprimerswereshowninFig. 1.Eh-F3-SERandEh-B3-SERprimerswereusedasouterprimersforfirstroundofnPCRamplificationwhichcouldgenerateampliconwithsize223 bp.Meanwhile,theprimerpairusedforconventionalPCR,secondroundofnPCRandqPCRamplificationwereadaptedfromF2regionofEh-FIP-SERasforwardprimerandB2regionofEh-BIP-SERasreverseprimer;whichcouldgenerateampliconwithsize175 bp.TheprimersusedforLAMPamplificationthatcoupledwithLFD,particularlyEh-BIP-SERandEh-LB-SERweresubjectedformodificationusinghapten-labellingat5′endoftheoligonucleotidesequence.Fluoresceinwaslabelledonthe5′endofEh-BIP-SERwhileEh-LB-SERwerelabelledwithbiotin.AllthesequenceofprimersusedinthisstudywerelistedinTable 2.ThespecificityoftheseprimerswasverifiedempiricallyusingDNAisolatedfromE.histolytica,E.dispar,E.moshkovskiiLaredoand75otherpathogensasshowninTable 1beforethecomparisonofanalyticalsensitivityamongtheassayswasconducted. Fig.1PrimerregionsonSREHPgenesequence(GenBankaccessionno.M80910.1).Eh-FIP-SERprimerisformedwithF1CandEh-F2whileEh-BIP-SERprimerisformedwithB1CandEh-B2FullsizeimageTable2OligonucleotidesequencesusedinthisstudyFullsizetableFormulationofLAMP,conventionalPCR,nPCRandqPCRassaysLAMPTheouterprimertoinnerprimerratiowasoptimisedandtheconcentrationofprimerswereoptimalwith2 μMofeachforwardinnerprimer(Eh-FIP-SER)andbackwardinnerprimer(Eh-BIP-SER),0.167 μMofeachforwardouterprimer(Eh-F3-SER)andbackwardouterprimer(Eh-B3-SER),and0.333 μMofbackwardloopprimer(Eh-LB-SER).TheconcentrationsofLAMPcomponentssuchasdNTPsmix,betaine,MgSO4,andBstDNApolymerasewereoptimisedanddeterminedempirically.Thereactionwascarriedoutwithafinalvolumeof30 μLreactionmixturecontaining1 × isothermalamplificationbuffer[20 mMofTris-HCl(pH 8.8),50 mMofKCl,10 mMof(NH4)2SO4,2 mMofMgSO4,0.1%ofTween20](NewEnglandBiolabs,Massachusetts,USA),0.6 mMofdNTPmix(ThermoFisherScientific,USA),0.8 Mofbetaine(Sigma,Missouri,USA),supplementary6 mMofMgSO4(NewEnglandBiolabs,Massachusetts,USA),16 UofBst2.0WarmStartDNApolymerase(NewEnglandBiolabs,Massachusetts,USA)and2 μLofDNAtemplate.Thereactionwascarriedoutat63 °Cfor60 minfollowedbyterminationat80 °Cfor5 min.TheLAMPproductwassubjectedtoagarosegelelectrophoresis,LFDandcalcein-manganesedyeforpost-LAMPanalysis.ConventionalPCREh-F2andEh-B2primerswiththeconcentrationof1 μMeachwereusedforconventionalPCRamplification.Theamplificationwascarriedoutinafinalvolumeof20 μLcontaining1 × PCRbuffer,2.5 mMofMgCl2(ThermoFischerScientific,Massachusetts,USA),0.16 mMofdNTPsmix(ThermoFischerScientific,Massachusetts,USA),1 UofTaqDNApolymerase(ThermoFischerScientific,Massachusetts,USA)and2 μLofDNAtemplate.PCRreactionwasperformedwithinitialdenaturationat95 °Cfor5 min,followedby35 cyclesof95 °Cfor30 s,56 °Cfor30 sand72 °Cfor30 s;andafinalextensionat72 °Cfor5 min.Theproductwassubjectedtogelelectrophoresisin2%agarosegelstainedwithGelStaindye(TransGenBiotechCo,Beijing),electrophoreticallyrununder100 Vfor60 minfollowedbyvisualisedusingChemiImage 5000analyser.nPCRThefirstroundofPCRwascarriedoutwithEh-F3-SERandEh-B3-SERprimerpairwiththeconcentrationof1 μMeach.Theamplificationwascarriedoutinafinalvolumeof20 μLcontaining1 × PCRbuffer,2.5 mMofMgCl2(ThermoFischerScientific,Massachusetts,USA),0.16 mMofdNTPsmix(ThermoFischerScientific,Massachusetts,USA),1 UofTaqDNApolymerase(ThermoFischerScientific,Massachusetts,USA)and2 μLofDNAtemplate.PCRreactionwasperformedwithinitialdenaturationat95 °Cfor5 min,followedby30 cyclesof95 °Cfor30 s,60 °Cfor30 sand72 °Cfor30 s;andafinalextensionat72 °Cfor5 min.Meanwhile,thesecondroundofPCRwascarriedoutwithEh-F2andEh-B2primerpairwiththeconcentrationof1 μMeach.ThereagentcompositionusedwassimilartothefirstroundPCR.Thereactionwasperformedwithinitialdenaturationat95 °Cfor5 min,followedby35 cyclesof95 °Cfor30 s,56 °Cfor30 sand72 °Cfor30 s;andafinalextensionat72 °Cfor5 min.Theproductwassubjectedtogelelectrophoresisin2%agarosegelstainedwithGelStaindye,electrophoreticallyrununder100 Vfor60 minthenvisualisedusingChemiImage 5000analyser.qPCRSimilartoconventionalPCRandsecondroundreactionofnPCR,theqPCRreactionwascarriedoutwithEh-F2andEh-B2primerpairwiththeconcentrationof1 μMeach.Theamplificationwascarriedoutinafinalvolumeof25 μLcontaining1 × QuantiFastSYBRGreenPCRMasterMixand2 μLofDNAtemplate.Thereactionwasperformedwiththermalcyclingconditionofinitialdenaturationat95 °Cfor5 min,followedby40 cyclesof95 °Cfor20 sand56 °Cfor30 s.Meltingcurveanalysiswasperformedataslowincreasefrom65 °Cto95 °Cwithaspeedof0.5 °Cper5 s.Baselinethresholdforthepost-amplificationanalysiswassetat50relativefluorescenceunits(RFU)andanyquantitationcycle(Cq)valuebeloworequalto38isconsideredpositive.Preparationofpost-LAMPanalysisConstructionofLFDGoldnanoparticle(GNP)wasusedassignalgeneratorforLFDinthisstudy.Thebio-conjugationofGNPswithFITCIgG1monoclonalantibodyandthepreparationofLFDwascarriedoutasdescribedbyFooetal.[21]withsomemodification.TheLFDstripwithsizeof5 mm × 77 mmcomposedwithbufferapplicationpad,goldconjugatepad,nitrocellulosemembraneandanabsorbentpadasshowninFig. 2.TheLFDwasaffixedwith1 μggoatanti-mouseIgGsecondaryantibodyaschromatographycontrolline(CCL)and2 μgstreptavidinastestline(TL)followedbyblocktheuncoatednitrocellulosesurfacewithblockingbuffer[mixtureof0.2%WBR,0.05%tritonX-100and2 mMphosphatebuffer(PB)].ThegoldconjugatepadwasmadewithfunctionalisedconjugatedGNPssuspension[5OD522goldconjugatesuspendedin2 mM PBcontaining20%(w/v)sucrose,0.01%(v/v)PVAand0.01%(v/v)Tween-20]indry-reagentformatasdescribedbyFooetal.[21]. Fig.2SchematicdiagramoflateralflowdipstickstripFullsizeimagePreparationofcalcein-manganesedyeCalcein-manganesedyewasmadeof500 μMcalceinthatdissolvedindimethylsulfoxide(Merck,USA)and10 mMMnCl2thatdissolvedinnuclease-freewater.Only1 μLofcalcein-manganesedyerequiredforeveryreaction.PrincipleofLFDTheLFDwasformulatedtospecificallyrecogniseitsdouble-labelleddouble-strandedDNAampliconthroughthebindingofthebiotinlabelledonthe5′endofampliconstothestreptavidinonnitrocellulosemembraneofLFD.Testlinerequiredamplifieddouble-labelleddouble-strandedampliconsastheconnectionbridgetogeneratesignalthatrepresentthepresence/absenceofthetargetDNA.Thedouble-labelleddouble-strandedamplicons(LAMPproduct)forTLwerelabelledwithFAMat5′endsynthesisedbyinnerprimerswhereasanother5′endthatsynthesisedbyloopprimerwerelabelledwithbiotinFig. 3.StreptavidinonTLformedprotein-ligandbindingwithbiotinondoubled-labelledamplicons.Meanwhile,CCLaffixedwithgoatanti-mouseIgGsecondaryantibodyformedprotein–ligandaffinitybindingwithmousemonoclonalFITCIgG1antibodywhichconjugatedongoldnanoparticles. Fig.3Schematicillustrationoftheformationofdouble-labelledamplicon(LAMPproduct)worksastheanalyteforLFDdetectionFullsizeimageFigure 4illustratedaschematicprincipleoftheLAMPproductcapturedbystreptavidinondetectionpadofLFD.Streptavidinaffixedonthedetectionregionimmobilisedtheampliconsthroughprotein-ligandbondingformedwiththebiotinlabelledontheamplicons.Thedouble-strandedampliconswereformedbytheLAMPinnerprimersequencesandloopprimersequences.Therefore,theother5′endofampliconwaspresentedwithFAMthatboundwithgoatanti-mouseantibodyconjugatedongoldnanoparticles.ThepresenceofpinkishredlineonthedetectionregionshowedthecompletionofhybridisationsandwichamongtheconjugatedGNPs,LAMPproductandstreptavidinwherebyinterpretedaspositiveresult. Fig.4SchematicillustrationoftheprincipleofLFD.TheillustrationshowstheLAMPproductisimmobilisedonthemembranebystreptavidin,followedwithsignalgenerationbyconjugatedGNPsthatbindonfluoresceinFullsizeimagePost-LAMPanalysisPost-LAMPanalysiswascarriedoutin3differenttechniquesnamelyagarosegelelectrophoresis,LFDandcalcein-manganesedye.LAMPproductwassubjectedtogelelectrophoresisin2.5%agarosegelstainedwithGelStaindye,electrophoreticallyrununder100 Vfor80 minfollowedbyvisualisedusingChemiImage 5000analyser.Thepresentofladder-likebandspatternontheagarosegelindicatedpositiveamplification.DetectionofLAMPproductusingLFDwassimilartoapreviousstudy[21].Theamplifiedproductwithanamountof4 μLwasmixedwith16 μLofrunningbuffer[1 × PBSand1%(v/v)Tween-20]andthemixturewasdroppedontothedetectionregion.TheLFDwasthenplacedverticallyfollowedbydippingthebufferapplicationpadinto250 μLofrunningbuffer.Theresultcouldbevisualisedwithunaidedeyeswithin10to15 minontheLFDnitrocellulosemembrane.TheformationofreddottedlineonTLindicatedpositiveresultwhiletheabsentofTLindicatednegativeresult.CCLontheLFDservedasproceduralandoperationalcontrolforeveryLAMPproductanalysis.ThelineformationonCCLindicatedeffectivenessofsignalgenerator,thefunctionalisedGNPswhiletheabsentofCCLindicatedfalsenegativeresult.AnalysisofLAMPproductusingcalcein-manganesedyewascarriedoutbymixing1 μLofcalcein-manganesedyeintothe30 μLofLAMPreagentmixbeforeamplification.WiththeaidofUVlight,tubethatturnedfluorescentgreenwasconsideredpositivewhilenegativeamplificationremainedasdimgreen.AnalyticalspecificityofassaysTheanalyticalspecificityoftheprimersusedforLAMP,conventionalPCR,nPCRandqPCRapplicationweredeterminedusingDNAisolatedfromE.histolytica,E.dispar,E.moshkovskiiLaredoand75otherpathogensaslistedinTable 1.Toensureitsspecificitywascomprehensivelyinvestigated,thisstudywasconductedseparatelyonLAMP,conventionalPCR,nPCRandqPCR.EvaluationofassaysperformanceAllthe4amplificationassayswereoptimisedinthisstudy.TheperformanceoftheassayswasevaluatedbasedontheiranalyticalsensitivityintermofLoDusing10-folddilutedE.histolyticatrophozoites.Thedilutedtrophozoitesinarangeof106to10− 4trophozoiteswerespikedinto200 mgofstoolsamplesandleftfor1 hatambienttemperaturepriortoDNAisolation.ExtractedDNAsfromthespikedstoolsamplesinarangeof106to10− 3trophozoiteswereusedforPCRwhileLoDforLAMPamplificationwasdeterminedusingtrophozoitesrangeupto10− 4.Alltheevaluationtestswereperformedintriplicate.ResultsDevelopmentandoptimisationofLAMPassayInvestigationonLAMPprimerratiowasprioritisedbeforeotherLAMPcomponentswereoptimisedasincompatibleratioforinnerandouterLAMPprimersmayaffecttheamplificationsensitivity[22].Theouterprimerstoinnerprimersratiowasdeterminedusingtwoseparatedexperiments,namelyinnerprimersconcentrationoptimisationandouterprimersconcentrationoptimisation.Theoptimisationforinnerprimersconcentrationwasconductedbyalteringtheconcentrationsofinnerprimersfrom0.33to2.67 μMwiththeouterprimerskeptat0.167 μM.Ontheotherhand,theconcentrationofouterprimerswasoptimisedinarangeof0.083to0.67 μMwiththeinnerprimerskeptat1.33 μM.Theoptimalinnerprimersconcentrationwasfoundtobe2.0 μM(Fig. 5a),whiletheoptimalouterprimersconcentrationwasrecordedas0.167 μM(Fig. 5b). Fig.5OptimisationofLAMP(a)innerprimersconcentrationand(b)outerprimersconcentration.L,100 bpDNAladder;B,blank(noDNAtemplatecontrol);N,negativecontrol;1–16,primerconcentrationsinμM:lane1,0.33;lane2,0.67;lane3,1.0;lane4,1.33;lane5,1.67;lane6,2.0;lane7,2.33;lane8,2.67;lane9,0.083;lane10,0.10;lane11,0.117;lane12,0.133;lane13,0.167;lane14,0.25;lane15,0.33;lane16,0.67.Theselectedoptimuminnerprimersconcentrationwas2.0 μMwhile0.167 μMwasselectedasoptimumouterprimersconcentrationFullsizeimageLAMPcomponentswereoptimisedtoensuretheefficiencyofamplification.Parametersincludingincubationtemperature,concentrationsofbetaine,MgSO4,dNTPmix,andBstDNApolymerasewereempiricallydetermined.OptimisationofLAMPparametersdidnotsignificantlyimprovetheefficiencyofamplification.However,studyonthebetaineconcentrationshowedexcessofbetainecoulddetertheefficiencyofamplification.Figure 6showedtheoutcomeofLAMPamplificationperformedwith3differentconcentrations(1.6 M,0.8 Mand0.4 M).LAMPreactionperformedwithlowerbetaineconcentration(0.4 M)couldtoleratefalsepositiveresultwhileexcessbetaine(1.6 M)coulddeteramplificationefficiency.LAMPamplificationconductedwithoutDNAtemplateandwithnon-E.histolyticaDNAcouldgeneratefalsepositiveresultwhen0.4 Mbetainewasincorporatedintothereaction.Althoughamplificationwith1.6 Mbetainedoesnotgeneratefalsepositiveresultonnegativecontrolandnon-targetcontrol,theproductintensityofpositivecontrolonagarosegelwasfaint.Therefore,0.8 Mwasselectedastheoptimumconcentrationforbetaine.Theoptimised30 μLofLAMPreactionmixwasconcludedasoptimalwith1 × LAMPamplificationbuffer,0.8 Mofbetaine,6 mMofMgSO4,0.6 mMofdNTPmixand16 UofBstDNApolymerasewithincubationtemperatureof63 °C. Fig.6EffectofbetaineinLAMPamplification.L,100 bpplusDNAladder;B,blank(noDNAtemplatecontrol);1,E.histolytica;2,E.dispar.Theconcentrationofbetaineusedwere:setA,1.6 M;setB,0.8 M;setC,0.4 M.Theselectedoptimumbetaineconcentrationwas0.8 MFullsizeimageAnalyticalperformancesofLAMP,conventionalPCR,nPCRandqPCRassaysAnalyticalspecificityTheprimersanalyticalspecificityforLAMP,conventionalPCR,nPCRandqPCRrespectiveapplicationusingDNAisolatedfromE.histolytica,E.dispar,E.moshkovskiiLaredoand75otherpathogensrevealed100%specificity.TheanalyticalspecificityoftheprimersusedfortheirrespectiveamplificationsissummarisedinTable 1.AnalyticalsensitivityDetectionlimitofthe4assayswereinvestigatedusingDNAsisolatedfrom10-folddilutedtrophozoitesthatwerespikedintostoolsamples.ThosetrophozoiteswerespikedintostoolsamplesobtainedfromhealthyindividualstosimulateisolationofDNAfromstoolofinfectedindividual.TheextractedDNAsinarangeof106to10− 3trophozoiteswereusedforPCRapplicationswhileLAMPamplificationwastestedusingupto10− 4trophozoitesduetoitsexcellentamplificationefficacy.TheLoDforconventionalPCRwasfoundtobe1000trophozoitesasshowninFig. 7awhilenPCRrecorded10-foldmoresensitiveLoDasampliconwasstillobservablewhentestedwithDNAextractedfrom100trophozoites(Fig. 7b).Asonlycurvegoesabove50RFUwith≤38Cqvaluewillbeinterpretedaspositive(seemethods),theanalyticalsensitivityofqPCRwasfoundtobesimilartonPCR,whereinwasrecordedas100trophozoites(Fig. 7c).TheanalyticalsensitivityoftheLAMPassayconductedusing3differentpost-LAMPanalysesrecordedsimilarLoDinwhichagarosegelelectrophoresis(Fig. 8a),calcein-manganesedye(Fig. 8b)andLFD(Fig. 8c)techniquessimilarlyrecorded1trophozoiteasLoD. Fig.7Spikedstoolanalyticalsensitivityon(a)conventionalPCR,(b)nPCRand(c)qPCRusing10-folddilutionsofE.histolyticatrophozoites.L,100 bpDNAladder;N,negativecontrol;1–10,10-folddilutionoftrophozoites:lane1,106;lane2,105;lane3,104;lane4,103;lane5,102;lane6,10;lane7,1;lane8,0.1;lane9,10− 2;lane10,10− 3.PCRcoulddetectupto1000trophozoiteswhilenPCRandqPCRcoulddetectupto100trophozoitesFullsizeimageFig.8SpikedstoolanalyticalsensitivityonLAMPusing10-folddilutionsofE.histolyticatrophozoitesandanalysedusing(a)agarosegelelectrophoresis,(b)calcein-manganesedyeand(c)LFD.L,100 bpDNAladder;N,negativecontrol;1–10,10-folddilutionoftrophozoites:lane1,106;lane2,105;lane3,104;lane4,103;lane5,102;lane6,10;lane7,1;lane8,0.1;lane9,10− 2;lane10,10− 3;lane11,10− 4.Allthe3post-LAMPanalysesrecorded1trophozoiteasLoDFullsizeimageDiscussionPCRhasalwaysbeentheDNA-baseddetectionmethodofchoiceforidentificationofmedicallyimportantpathogen.Asforamoebiasis,thismethodhasbeenendorsedbyWHOfordetectionofE.histolytica.ThistechnologyhasbeenreportedtodetectE.histolyticainvariousclinicalspecimensandcoulddifferentiateitfromothermorphologicallyindistinguishablenon-pathogenicEntamoebaspecies[23,24].However,thepresenceofamplificationinhibitorshashinderedPCRapplicationfordetectionofE.histolyticainfaecalsamples.Examplesoftheamplificationinhibitorsincludebilirubins,bilesalts,hemeandcarbohydratesandsuchmoleculecomplexescouldbeco-extractedalongwiththepathogenDNAinstoolsamples[25].LAMPamplificationthatusessingletemperaturetoamplifytargetgenewasdescribedtobemorerobustandlessaffectedbyinhibitoryagentsfoundinclinicalsamples[26].ThisfindingwasfurtherverifiedwhenEngkuNurSyafirahetal.[16]foundreductionofamplificationsensitivityonPCRwhileLAMPcouldretainitsefficacywhentestedwithDNAisolatedfromspikedstoolsamples.TherobustnessofLAMPhasencouragedseveraldevelopmentsofdiagnosticassays.Inregardstodiagnosisofamoebiasis,Liangetal.[18],RiveraandOng[19],Singhetal.[20]andFooetal.[21]hadshownthepotentialofLAMPintermsofefficiencyandapplicabilityfordetectionofthepathogen.ThisstudywasconductedtofurthercomparetheLAMPamplificationperformancewithconventionalPCR,nPCRandqPCR.Empiricalanalysisonthedesignedprimersusingcross-amplificationanalysisandprimerconcentrationoptimisationwereconductedtomaximisetheamplificationefficacy.DuringLAMPamplification,theinnerprimerF2andB2regionswouldfirstbindandpolymerisetheircomplementarynucleotidesequencesbeforeadherenceofouterprimersF3andB3followedbyexecutingstranddisplacementactivity.ThismechanismshowedtheLAMPinnerprimersplayedanimportantroleindeterminingthespecificityofLAMPamplification.Therefore,conventionalPCR,andqPCRamplificationsweresolelyconductedusingF2andB2regionsofLAMPinnerprimerswhilenPCRwasconductedwithadditionalLAMPouterprimers.Primersspecificityverificationshowedgenerationofexpectedproductsizesonagarosegelelectrophoresiswhiletheircross-amplificationwithotherEntamoebaspp.andnon-EntamoebaDNAsalsoshowedexpectedspecificities.Cross-amplificationwasconductedonLAMPprimersetusingLAMPamplificationandtheresultsobtainedshowedexpectedoutcomes.DuringtheLAMPassaydevelopment,theconcentrationofinnerandouterprimerwasinvestigatedtoavoiddisproportionprimersratiothatcouldaffecttheamplificationefficiency[22].ThisoptimisationrevealedincrementofinnerprimersconcentrationcouldsignificantlyincreasetheamplificationyieldwhiletheinvolvementofouterprimersinLAMPamplificationdidnotaltertheampliconyield.Besides,theLAMPouterprimersthatwasonlyinvolvedindisplacingtheinnerprimertoallowtheformationofdumbbell-likeDNAstructurewasdevoidwithF3andB3regions.Therefore,thestrategyofhavingfluoresceinlabelledonbackwardinnerprimerandbiotinonloopprimerwasrelevantandapplicable.DuringthedevelopmentofLAMPassayinthisstudy,betainewasfoundtobeanessentialcomponentthatcouldenhancethespecificityofLAMPamplification.Theoptimisationstudyconductedwithlowerbetaineconcentrations(0.4 M)hasfacilitatedtheformationoffalsenegativeresults.However,excessincrementofbetaine(1.6 M)coulddistorttheamplificationefficiencyandreducetheassaysensitivity.Therefore,betaineoptimisationiscrucialineveryLAMPassaydevelopmenttoeliminateunspecificamplificationandyetretainitsamplificationefficiency.TheapplicationofdirectvisualidentificationonLAMPproductusingintercalatingfluorescentdyehasbeenreportedtobesensitiveandspecific[27,28].Anin-housecalcein-manganesedyewasusedaspost-LAMPanalysisfordirectvisualidentificationonLAMPproductinthisstudy.Thisfluorescentindicatormadeuseofpyrophosphate,aLAMPamplificationby-product,togeneratefluorescentsignalbyallowingmanganeseiontoformcomplexmoleculewithpyrophosphateandleavingfreecalceintoirradiatethefluorescenceunderUVlight.Lateralflowdipstick-basedassayisafeasiblediagnosticplatformbecauseitissimpletoperform,producerapidvisualresult,amendableformass-production,relativelycheaptoproduce,doesnotrequireequipmentforresultinterpretationandportable[3].Thein-housedry-reagentLFDcoulddetecttargetnucleicacidbycapturingthehaptenlabelledampliconandgeneratedvisualsignalusingfunctionalisedGNPs[21].Incorporationofdry-reagenttechnologyonLFDforefficacypreservationandstabilityenhancementhasexpandeditsusabilityandmadeitoperatorfriendly.Therefore,these2methodswereselectedasalternativepost-LAMPanalysesforsensitivitycomparisonwithagarosegelelectrophoresis.ThisstudyrevealedthesensitivityofPCRamplificationplatformwasnotcomparabletoLAMPregardlessofitsamplificationandampliconanalysisinnovations.AlthoughtheadditionofamplificationcyclesasdemonstratedbynPCRandqPCRcouldpromoteyieldincrement,excessiveamplificationcyclesinasinglereactionwouldriskfaultyprimingthatcouldgeneratefalsepositiveresult[4].Besides,theenhancementstilllackedsensitivityasLAMPcoulddetectDNAwithconcentrationof100-foldlowercomparedtonPCRandqPCR.Meanwhile,thesimilarLoDrecordedbythe3LAMPpost-amplificationanalysisdemonstratedreproducibilityofLAMPamplification.Theanalyticalsensitivityconsistencydespitebeingcoupledwithdifferentpost-amplificationanalysismethodsjustifiedthecompatibilityandrobustnessofLAMPasanalternativenucleicacidamplificationtesttoPCR.ThestrengthofLFDandcalcein-manganesedyeusedduringpost-LAMPanalysisalsoupheldthepotentialofLAMPinbecominganequipment-freediagnosticassaythatcompliedtoASSUREDprinciple.ThisprinciplewhichstandsforAffordable,Sensitive,Specific,User-friendly,Rapidandrobust,Equipment-freeandDeliverablewasdeemedrelevantandindemandforbetterdiseasecontrolinthedevelopingcountrieswithlowresourcesetting[29].DespiteLAMPoutstandingperformance,thetechnologywaslessfavourableformultiplexing,whichinvolvedtoleratingamplificationofmultipletargetsinasinglereaction.Moreover,thetypicalladder-likepatternofLAMPproduct,producedbymixtureofdumbbell-likestructureswithvariousstemlengthareindistinguishableundergelelectrophoresisandintercalatingfluorescentdye.ThissetbackwasresolvedbyNajianetal.[14]whodevelopedaduplexLAMPassaywiththeaidofLFD;followedbytheexpansionofthetechniquetoatriplexLAMPassaybyFooetal.[21].TheincorporationofinternalamplificationcontrolintotheseassaystoruleoutfalsenegativeresultcausedbyamplificationinhibitorshasfurtherenhancedLAMPfeasibilityasanalternativemoleculartechniquefordetectionofpathogens.ConclusionTheanalyticalsensitivitycomparisonamongtheconventionalPCR,nPCR,qPCRandLAMPrevealsthatLAMPoutperformedtherestintermsofLoDandamplificationtime.Meanwhile,allthe3post-LAMPanalysesappearedtobesimilarindetectionsensitivities.Hence,LAMPisarelevantalternativeDNA-basedamplificationplatformforsensitiveandspecificdetectionofpathogens. Availabilityofdataandmaterials Alldatageneratedoranalysedduringthisstudyareincludedinthispublishedarticle. AbbreviationsBSA: Bovineserumalbumin Bst : Bacillusstearothermophilus CCL: Chromatographycontrolline dNTPs: Deoxynucleosidetriphosphates FITC: Fluoresceinisothiocyanate GNP: Goldnanoparticle LAMP: Loop-mediatedisothermalamplification LFD: Lateralflowdipstick LoD: Limitofdetection nPCR: Nestedpolymerasechainreaction PVA: Polyvinylalcohol PVP: Polyvinylpyrrolidone qPCR: Real-timepolymerasechainreaction RFU: Relativefluorescenceunits SREHP : Serine-richEntamoebahistolyticaprotein Taq : Thermusaquaticus TL: Testline WBR: Westernblockingreagent ReferencesChangD,TramK,LiB,FengQ,ShenZ,LeeCH,SalenaBJ,LiY.DetectionofDNAampliconsofpolymerasechainreactionusinglitmustest.SciRep.2017;7(1):3110.Article  GoogleScholar  LawJW,AbMutalibNS,ChanKG,LeeLH.Rapidmethodsforthedetectionoffoodbornebacterialpathogens:principles,applications,advantagesandlimitations.FrontMicrobiol.2014;5:770.PubMed  GoogleScholar  YagerP,DomingoGJ,GerdesJ.Point-of-carediagnosticsforglobalhealth.AnnuRevBiomedEng.2008;10:107–44.CAS  Article  GoogleScholar  LorenzTC.Polymerasechainreaction:basicprotocolplustroubleshootingandoptimizationstrategies.JVisExp.2012;63:e3998. 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DownloadreferencesAcknowledgementsTheauthorswishtothanktheDirectorGeneralofHealthMalaysiaforhispermissiontopublishthisarticle.SpecialthanksarealsodedicatedtoProfessorGrahamC.Clark(LondonSchoolofHygieneandTropicalMedicine,London,UK)forprovidingE.moshkovskiiDNAandDr.AlfonsoOlivos-Garcia(DepartamentodeMedicinaExperimental,FacultaddeMedicina,UniversidadNacionalAutónomadeMéxico,Mexico)forprovidingE.histolyticaandE.dispartrophozoites.FundingThisstudywasfundedbyUniversitiSainsMalaysia(ResearchUniversityGrants1001/PPSK/813044and1001/PPSP/812144)andMinistryofHigherEducationMalaysia(PrototypeDevelopmentResearchGrantScheme 203/PPSP/6740015).Allotheraspectsoftheprojectincludingdesignofthestudy,experimentalwork,analysisandinterpretationofdata,andwritingofthemanuscriptwasexecutedbytheauthors.AuthorinformationAuthorsandAffiliationsAcarologyUnit,InfectiousDiseaseResearchCentre,InstituteforMedicalResearch,MinistryofHealthMalaysia,NationalInstitutesofHealthComplex,BandarSetiaAlam,40170,ShahAlam,Selangor,MalaysiaPhiawChongFoo & MarianaAhamadDepartmentofMedicalMicrobiologyandParasitology,SchoolofMedicalSciences,UniversitiSainsMalaysia,HealthCampus,16150,KubangKerian,Kelantan,MalaysiaPhiawChongFoo, A.B.NurulNajian & ChanYeanYeanBacteriologyUnit,InfectiousDiseaseResearchCentre,InstituteforMedicalResearch,MinistryofHealthMalaysia,NationalInstitutesofHealthComplex,BandarSetiaAlam,40170,ShahAlam,Selangor,MalaysiaA.B.NurulNajianInstituteforResearchinMolecularMedicine,UniversitiSainsMalaysia,HealthCampus,16150,KubangKerian,Kelantan,MalaysiaNuraminA.MuhamadFacultyofVeterinaryMedicine,UniversitiMalaysiaKelantan,CityCampus,PengkalanChepa,Lockedbag36,16100,KotaBharu,Kelantan,MalaysiaMaizanMohamedHospitalUSM,UniversitiSainsMalaysia,HealthCampus,16150,KubangKerian,Kelantan,MalaysiaChanYeanYeanSchoolofHealthSciences,UniversitiSainsMalaysia,HealthCampus,16150,KubangKerian,Kelantan,MalaysiaBoonHuatLimAuthorsPhiawChongFooViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarA.B.NurulNajianViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarNuraminA.MuhamadViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarMarianaAhamadViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarMaizanMohamedViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarChanYeanYeanViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarBoonHuatLimViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarContributionsPC,MA,MM,CYandBHconceptualisedanddesignedtheexperiment.PC,NNandMNexecutedtheexperiments.PCcompiledtheresultsandpreparedthefirstdraftofthemanuscript.MM,CYandBHrevisedthefirstdraftofthemanuscript.PCandMAeditedandfinalisedthemanuscript.Allauthorsreadandapprovedthefinalmanuscript.CorrespondingauthorCorrespondenceto PhiawChongFoo.Ethicsdeclarations Ethicsapprovalandconsenttoparticipate Notapplicable. 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ReprintsandPermissionsAboutthisarticleCitethisarticleFoo,P.C.,NurulNajian,A.B.,Muhamad,N.A.etal.Loop-mediatedisothermalamplification(LAMP)reactionasviablePCRsubstitutefordiagnosticapplications:acomparativeanalysisstudyofLAMP,conventionalPCR,nestedPCR(nPCR)andreal-timePCR(qPCR)basedonEntamoebahistolyticaDNAderivedfromfaecalsample. BMCBiotechnol20,34(2020).https://doi.org/10.1186/s12896-020-00629-8DownloadcitationReceived:02January2020Accepted:16June2020Published:22June2020DOI:https://doi.org/10.1186/s12896-020-00629-8SharethisarticleAnyoneyousharethefollowinglinkwithwillbeabletoreadthiscontent:GetshareablelinkSorry,ashareablelinkisnotcurrentlyavailableforthisarticle.Copytoclipboard ProvidedbytheSpringerNatureSharedItcontent-sharinginitiative KeywordsLoop-mediatedisothermalamplificationNestedPCRReal-timePCRLateralflowdipstickCalcein-manganesevisualizationLAMPanalyticalsensitivity DownloadPDF Advertisement BMCBiotechnology ISSN:1472-6750 Contactus Submissionenquiries:[email protected] Generalenquiries:[email protected]