基于CRISPR-Cas基因編輯技術結合等溫擴增技術快速檢測碳青霉烯酶的研究

基于CRISPR-Cas基因編輯技術結合等溫擴增技術快速檢測碳青霉烯酶的研究摘要：

碳青霉烯酶產生菌株的廣泛傳播已經成為抗菌藥物應用問題的重要挑戰(zhàn)。目前，CRISPR-Cas基因編輯技術和等溫擴增技術已經成為檢測菌株中碳青霉烯酶的一種新方法。本研究旨在結合這兩種技術，建立一種新型的快速檢測碳青霉烯酶的方法，該方法可以在30分鐘內完成檢測。首先，我們通過文獻調查了CRISPR-Cas基因編輯技術在基因組檢測中的應用，并分析了其優(yōu)勢和局限性。其次，我們介紹了等溫擴增技術及其與CRISPR-Cas基因編輯技術的結合在基因組檢測中的應用。最后，我們詳細闡述了在建立快速檢測碳青霉烯酶的方法過程中，如何將兩種技術用于檢測菌株中碳青霉烯酶的豐度和基因的存在。我們的研究結果表明，結合CRISPR-Cas基因編輯技術和等溫擴增技術，可以快速、準確地檢測出菌株中的碳青霉烯酶，這一技術具有廣泛的應用前景。

關鍵詞：CRISPR-Cas基因編輯技術；等溫擴增技術；碳青霉烯酶；基因組檢測；豐度檢測；基因存在檢測

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