基于CRISPR-Cas基因編輯技術(shù)結(jié)合等溫?cái)U(kuò)增技術(shù)快速檢測(cè)碳青霉烯酶的研究

基于CRISPR-Cas基因編輯技術(shù)結(jié)合等溫?cái)U(kuò)增技術(shù)快速檢測(cè)碳青霉烯酶的研究摘要：

碳青霉烯酶產(chǎn)生菌株的廣泛傳播已經(jīng)成為抗菌藥物應(yīng)用問(wèn)題的重要挑戰(zhàn)。目前，CRISPR-Cas基因編輯技術(shù)和等溫?cái)U(kuò)增技術(shù)已經(jīng)成為檢測(cè)菌株中碳青霉烯酶的一種新方法。本研究旨在結(jié)合這兩種技術(shù)，建立一種新型的快速檢測(cè)碳青霉烯酶的方法，該方法可以在30分鐘內(nèi)完成檢測(cè)。首先，我們通過(guò)文獻(xiàn)調(diào)查了CRISPR-Cas基因編輯技術(shù)在基因組檢測(cè)中的應(yīng)用，并分析了其優(yōu)勢(shì)和局限性。其次，我們介紹了等溫?cái)U(kuò)增技術(shù)及其與CRISPR-Cas基因編輯技術(shù)的結(jié)合在基因組檢測(cè)中的應(yīng)用。最后，我們?cè)敿?xì)闡述了在建立快速檢測(cè)碳青霉烯酶的方法過(guò)程中，如何將兩種技術(shù)用于檢測(cè)菌株中碳青霉烯酶的豐度和基因的存在。我們的研究結(jié)果表明，結(jié)合CRISPR-Cas基因編輯技術(shù)和等溫?cái)U(kuò)增技術(shù)，可以快速、準(zhǔn)確地檢測(cè)出菌株中的碳青霉烯酶，這一技術(shù)具有廣泛的應(yīng)用前景。

關(guān)鍵詞：CRISPR-Cas基因編輯技術(shù)；等溫?cái)U(kuò)增技術(shù)；碳青霉烯酶；基因組檢測(cè)；豐度檢測(cè)；基因存在檢測(cè)

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