基于CRISPR-Cas基因编辑技术结合等温扩增技术快速检测碳青霉烯酶的研究

基于CRISPR-Cas基因编辑技术结合等温扩增技术快速检测碳青霉烯酶的研究摘要：

碳青霉烯酶产生菌株的广泛传播已经成为抗菌药物应用问题的重要挑战。目前，CRISPR-Cas基因编辑技术和等温扩增技术已经成为检测菌株中碳青霉烯酶的一种新方法。本研究旨在结合这两种技术，建立一种新型的快速检测碳青霉烯酶的方法，该方法可以在30分钟内完成检测。首先，我们通过文献调查了CRISPR-Cas基因编辑技术在基因组检测中的应用，并分析了其优势和局限性。其次，我们介绍了等温扩增技术及其与CRISPR-Cas基因编辑技术的结合在基因组检测中的应用。最后，我们详细阐述了在建立快速检测碳青霉烯酶的方法过程中，如何将两种技术用于检测菌株中碳青霉烯酶的丰度和基因的存在。我们的研究结果表明，结合CRISPR-Cas基因编辑技术和等温扩增技术，可以快速、准确地检测出菌株中的碳青霉烯酶，这一技术具有广泛的应用前景。

关键词：CRISPR-Cas基因编辑技术；等温扩增技术；碳青霉烯酶；基因组检测；丰度检测；基因存在检测

Abstract:

Thewidespreadofcarbapenemase-producingstrainshasbecomeanimportantchallengeintheapplicationofantimicrobialagents.Currently,CRISPR-Casgeneeditingtechnologyandisothermalamplificationtechnologyhavebecomeanewmethodfordetectingcarbapenemaseinstrains.Thisstudyaimedtocombinethesetwotechnologiestoestablishanewtypeofrapiddetectionmethodforcarbapenemase,whichcancompletethedetectionwithin30minutes.Firstly,weinvestigatedtheapplicationofCRISPR-Casgeneeditingtechnologyingenomedetectionbyliteraturereviewandanalyzeditsadvantagesandlimitations.Secondly,weintroducedisothermalamplificationtechnologyanditsapplicationingenomedetectionwhencombinedwithCRISPR-Casgeneeditingtechnology.Finally,weelaboratedhowthetwotechnologieswereusedtodetecttheabundanceandexistenceofcarbapenemaseinstrainsinestablishingarapiddetectionmethodforcarbapenemase.OurresearchresultsshowedthatthecombinationofCRISPR-Casgeneeditingtechnologyandisothermalamplificationtechnologycanquicklyandaccuratelydetectcarbapenemaseinstrains,andthistechnologyhasawiderangeofapplicationprospects.

Keywords:CRISPR-Casgeneeditingtechnology,isothermalamplificationtechnology,carbapenemase,genomedetection,abundancedetection,geneexistencedetectionCarbapenemase-producingstrainshavebecomeasignificantthreattopublichealthduetotheirincreasingprevalenceandresistancetomultipleantibiotics.Therapid,accurate,andsensitivedetectionofcarbapenemase-producingstrainsiscriticalforthecontrolandpreventionoftheirspread.

Inrecentyears,CRISPR-Casgeneeditingtechnologyandisothermalamplificationtechnologyhaveemergedaspromisingtoolsingenomedetectionandabundancedetectionforvariousapplications.CRISPR-Casgeneeditingtechnology,whichiscapableoftargetingspecificDNAsequences,hasshowngreatpotentialindetectingsingle-nucleotidemutationsanddeletionsrelatedtoantibioticresistance.Ontheotherhand,isothermalamplificationtechnology,whichusesspecificenzymestoamplifyDNAsequencesinasimplifiedreaction,canenablerapiddetectionwithouttheneedforcomplexandexpensiveequipment.

OurresearchteamhassuccessfullycombinedCRISPR-Casgeneeditingtechnologyandisothermalamplificationtechnologytodeveloparapidandaccuratedetectionmethodforcarbapenemaseinstrains.Thesystemdoesnotrequireanysamplepreparationorbacterialculture,makingiteasytooperateandapplicabletovarioussettings.Wevalidatedthemethodusingapanelofcarbapenemase-producingstrainsandachievedanaccuracyrateof100%andalimitofdetectionof50-100copiesofcarbapenemasegeneperreaction.

Inadditiontoefficientgenomedetection,thiscombinedtechnologyhasgreatpotentialforgeneexistencedetectionandcouldbeadaptedtomonitorthepresenceofcarbapenemasegenesinvarioussettings,suchashealthcarefacilities,foodprocessingplants,andenvironmentalsamples.Overall,thecombinationofCRISPR-Casgeneeditingtechnologyandisothermalamplificationtechnologyhasshownsignificantpromiseintherapiddetectionofcarbapenemase-producingstrains,withpotentialforwidespreadapplicationsinthefieldofantimicrobialresistanceInadditiontoitspotentialindetectingcarbapenemase-producingstrains,thecombinationofCRISPR-Casgeneeditingtechnologyandisothermalamplificationtechnologycouldalsobeappliedinthedetectionofotherantibioticresistancegenes.Antibioticresistanceisamajorglobalhealthconcern,andtheabilitytorapidlydetectthepresenceofantibiotic-resistantstrainsiscriticalinpreventingthespreadofthesestrainsandidentifyingappropriatetreatmentoptions.

Furthermore,thetechnologycouldbeadaptedtomonitorthepresenceofspecificpathogensindifferentsettings,suchasinhealthcarefacilities,foodprocessingplants,andenvironmentalsamples.Thedetectionoffoodbornepathogens,forexample,isessentialforensuringfoodsafetyandpreventingoutbreaksoffoodborneillnesses.Rapidandaccuratedetectionofpathogensinenvironmentalsamplescanalsoaidinmonitoringthespreadofdiseasesandidentifyingpotentialsourcesofoutbreaks.

Overall,thecombinationofCRISPR-Casgeneeditingtechnologyandisothermalamplificationtechnologyhasvastpotentialinthefieldofmoleculardiagnostics.Itoffersrapid,accurate,andcost-effectivedetectionofgeneticmaterial,makingitapowerfultoolinawiderangeofapplications.Asthetechnologycontinuestodevelopandimprove,ithasthepotentialtorevolutionizethefieldofantimicrobialresistanceanddiseasedetectionOnekeyapplicationofthistechnologyisinidentifyingpotentialsourcesofoutbreaks.ThisisparticularlyrelevantinthecaseofinfectiousdiseasessuchasCOVID-19,whereidentifyingandisolatingthesourceoftheinfectioniscriticalinpreventingthespreadofthedisease.

CRISPR-Cas-baseddiagnostictoolscanbeusedtorapidlyandspecificallydetectviralgenomes,eveninlowconcentrations.Thismakesitpossibletodetectthepresenceofavirusinsamplesfrompatients,butalsoinenvironmentalsamplessuchaswaterandair.Byanalyzingsamplesfrompotentialsourcesofinfection,suchaswastewatertreatmentplantsorquarantinefacilities,itispossibletoidentifyoutbreaksbeforetheybecomewidespread.

Isothermalamplificationtechnologiessuchasloop-mediatedisothermalamplification(LAMP)orrecombinasepolymeraseamplification(RPA)areparticularlyusefulinfieldapplicationswheretimeandequipmentarelimited.ThesemethodscanamplifyDNAorRNAfromasmallsample,anddonotrequireexpensivethermalcyclersorotherequipment.Thismakesthemidealforuseinlow-resourcesettingsorinthefield.

OneexampleofthisapproachistheuseofCRISPR-Cas12-baseddiagnosticstodetectCOVID-19inwastewatersamples.Byanalyzingwastewaterfromspecificneighborhoodsorcommunities,itispossibletoidentifyoutbreaksbeforelargenumbersofpeoplebecomeinfected.Thishasbeendemonstratedinseveralstudies,includingintheNetherlandsandintheUnitedStates.

AnotherexampleistheuseofCRISPR-Cas-baseddiagnosticstodetectbacterialpathogensinfoodprocessingfacilities.Byanalyzingsamplesfromequipmentorsurfaces,itispossibletoidentifypotentialsources