等温扩增综述

970|Mol.BioSyst.,2014,10,970--1003Thisjournalis©TheRoyalSocietyofChemistry2014Citethis:Mol.BioSyst.,2014,10,970IsothermalamplifieddetectionofDNAandRNALeiYan,JieZhou,YueZheng,AdamS.Gamson,BenjaminT.Roembke,ShizukaNakayamaandHermanO.Sintim*Thisreviewhighlightsvariousmethodsthatcanbeusedforasensitivedetectionofnucleicacidswithoutusingthermalcyclingprocedures,asisdoneinPCRorLCR.Topicsincludedarenucleicacidsequence-basedamplification(NASBA),stranddisplacementamplification(SDA),loop-mediatedamplification(LAMP),Invaderassay,rollingcircleamplification(RCA),signalmediatedamplificationofRNAtechnology(SMART),helicase-dependentamplification(HDA),recombinasepolymeraseamplification(RPA),nickingendonucleasesignalamplification(NESA)andnickingendonucleaseassistednanoparticleactivation(NENNA),exonuclease-aidedtargetrecycling,JunctionorY-probes,splitDNAZymeanddeoxyribozymeamplificationstrategies,template-directedchemicalreactionsthatleadtoamplifiedsignals,non-covalentDNAcatalyticreactions,hybridizationchainreactions(HCR)anddetectionviatheself-assemblyofDNAprobestogivesupramolecularstructures.ThemajorityoftheseisothermalamplificationmethodscandetectDNAorRNAincomplexbiologicalmatricesandhavegreatpotentialforuseatpoint-of-care.1.0IntroductionSensingnucleicacidsequencesisvitalinmodernbiologyandmedicineandthefieldhasgrownexponentiallysincethe1990s,andislikelytocontinuetoincreaseinimportanceoverthenextfewdecades.Thedetectionofpathogenicbacteria/virusesandcancergenesinapatient’ssamplecanbecriticaltodecisionsthatimpactpatient’srecovery.Seminaldiscoveriesmadebyseveralgroupshavelinkedthesusceptibilitytodiseasessuchascancerorthedifferencesindrugmetabolismtosinglenucleotidepolymorphisms(SNPs)andshortinsertion/deletionvariations.1SeveralstudieshavealsoshownthatRNAplaysakeyroleincancerdevelopment.1–9HumanmiRNAgenesareoftenfoundnearcancer-associatedgenomicregionsandfragilesitesandsomemiRNAshavelowerlevelsofexpressionincancersamplescomparedtonormaltissues.2AbnormalexpressionofmiRNAshasbeenfoundincancersincludingchroniclymphocyticleukaemia,3colorectalneoplasia,4paediatricBurkittlymphoma,5lungcancer,6largecelllymphoma,7glio-blastoma,8andBcelllymphomas.9ProfilingmiRNAtoolsarethereforerequiredfortheunderstandingoftheexpressionpatternofmiRNA.Thedetectionofnucleicacidsequenceswithoutpriorpurificationandamplification,incellsorinbiologicalfluids,andevendirectlyinlivingorganismsishighlydesirable.Thedemandfordetectingspecificnucleicacidsequencesofdifferentlengthstoaidclinicaldiagnosisandforunderstandingfundamentalbiologyhasspurredthedevelop-mentofamyriadofDNA/RNAdetectiontechnologies.10Thepolymerasechainreaction(PCR)canbeusedtoexponentiallyamplifyafewcopiesoftargetgenestoconcentrationsthatcouldbereadilydetectedviaavarietyofmeans.DuetotheneedforaduplexmeltingstepduringthePCRcycle,whichrequiresheating,PCRisnotappropriateforthedetectionofnucleicacidsequencesinlivecells.Isothermalnucleicacidamplificationstrategies,especiallythosethatwouldworkatcellulartempera-tures(30–371C)couldaidstudiesthataimtounderstandthetemporalexpressionofgenesinlivecells.Forinvitroapplica-tions,theneedforathermalcyclinginstrumentorskilledtechniciantoconductthePCRassay(bothofwhichrequirecapitalinvestment)cansometimesbeprohibitiveforresource-poorsettings.TheseaforementionedlimitationsofPCR-basedDNA/RNAdetectionassayshavespurredthedevelopmentofvariousisothermaldetectionplatformsthatamplifythetarget,thesignalorboth.Theaimofthisreviewistohighlightsomeofthenucleicaciddetectionmethodologiesthatdonotrequirethermalcyclingprocedures,suchaspolymerasechainreactions(PCR)orligasechainreaction(LCR).WeacknowledgethatthisreviewdoesnotcovereveryisothermalDNA/RNAdetectiontechnologythathasbeenreportedtodate,asitwouldbeimpracticaltodosoinasinglereview,andtheomissionofanyamplifiedisothermaldetectionstrategydoesnotimplythattheomittedstrategyisdeemedtobeunexciting.Thereaderisreferredtootherexcellentreviewsonthistopic,suchasthereviewbyAsielloandBaeumner,whichfocusedonminiaturizedisothermalnucleicacidamplification11,andbyWilson,whichfocussedonDepartmentofChemistryandBiochemistry,UniversityofMaryland,CollegePark,MD20742,USA.E-mail:[e-mail]24thJuly2013,Accepted18thFebruary2014DOI:10.1039/c3mb70304e:31:02.ThisarticleislicensedunderaCreativeCommonsAttribution3.0UnportedLicence.ViewArticleOnlineViewJournal|ViewIssueThisjournalis©TheRoyalSocietyofChemistry2014Mol.BioSyst.,2014,10,970--1003|971goldnanoparticlesfordetection.12Also,thereviewsbyDong13aandLi13bonisothermalnucleicaciddetectionofmicroRNAarecomplementarytothisreview.132.0Isothermalnucleicacidsdetectionwithoutamplification2.01Insituhybridization(ISH)Insituhybridization(ISH)isanisothermalmethodthatwasdevelopedinthelate1960sbyPardueandGall.14ItwasintroducedintobacteriologybyGiovannoni,whowasthefirsttouseradioactivelylabeledoligonucleotideprobesforthedetec-tionofbacteria.15Radioactivelabelsweregraduallyreplacedbynon-isoto