基于RAA-CRISPR-Cas13a检测KPC型碳青霉烯酶基因方法的建立及评价  

Establishment and evaluation of a RAA-CRISPR-Cas13a method for detecting KPC carbapenemase genes

作　　者：曹亚玲 田原 范子豪 徐玲 高耀 张向颖[1] 任锋[1] Cao Yaling;Tian Yuan;Fan Zihao;Xu Ling;Gao Yao;Zhang Xiangying;Ren Feng(Beijing Youan Hospital/Beijing Institute of Hepatology,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学附属北京佑安医院、北京肝病研究所,北京100069

出　　处：《中华检验医学杂志》2024年第2期159-164,共6页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(81770611,82002243);北京自然科学基金和北京市教委联合资助重点项目(KZ202010025035);首都卫生发展科研专项重点攻关项目(SF2020-1-1151,SF2021-1G-2181,SF2022-1-2182);北京市临床诊疗技术示范应用与研究专项(Z191100006619096,Z191100006619097);北京市医院管理中心“青苗”计划专项(QML20201702);北京市医管局“登峰”人才计划(DFL20221503);高层次公共卫生技术人才建设项目(学科带头人-02-13)。

摘　　要：目的建立基于重组酶介导等温扩增技术(RAA)-规律成簇的间隔短回文重复序列及其相关蛋白(CRISPR-Cas13a)准确检测肺炎克雷伯菌碳青霉烯酶(KPC)型碳青霉烯酶基因的方法。方法收集2020—2021年北京市垂杨柳医院保存的25株产耐碳青霉烯类肺炎克雷伯菌(CRKP)临床分离株和5株碳青霉烯类敏感肺炎克雷伯菌(CSKP),提取菌株总DNA。设计检测KPC DNA特异性RAA引物和检测CRISPR RNA(crRNA),建立基于RAA-CRISPR-Cas13a技术快速准确检测KPC型碳青霉烯酶基因的方法。通过KPC质粒和临床样本菌株对方法进行评价,同时使用荧光定量聚合酶链反应(qPCR)方法进行检测,比较2种方法的检出率和一致性。结果本研究建立的RAA-CRISPR-Cas13a方法检测KPC质粒和样本的检测灵敏度均可达到1拷贝/μl,高于qPCR(101拷贝/μl)。RAA-CRISPR-Cas13a检测方法和qPCR方法均从30株临床菌株(包含25株CRKP菌株和5株CSKP菌株)中检出23株携带KPC基因,7株未检出KPC基因,25株CRKP菌株中KPC基因检出率为92%(23/25),2种检测方法阳性符合率为100%(23/23)。结论将RAA扩增技术结合CRISPR-Cas13a技术,建立了一种准确检测KPC型碳青霉烯酶基因的方法。该方法有助于精准的筛选产KPC型碳青霉烯酶的菌株。Objective To establish a rapid and accurate method for the detection of Klebsiella pneumoniae carbapenemase(KPC)carbapenemase gene based on recombinase aided amplification(RAA)-CRISPR-Cas13a(CRISPR-Cas13a)technology.Methods Twenty-five clinical isolates of carbapenem-resistant Klebsiella pneumoniae(CRKP)and five carbapenem-sensitive Klebsiella pneumoniae(CSKP)strains preserved in 2020-2021 in Beijing Chuiyangliu Hospital were randomly collected,and the total DNA samples of the strains was extracted.RAA primers specific for KPC DNA and CRISPR RNA(crRNA)were designed to establish a rapid and accurate method for the detection of KPC carbapenemase gene based on RAA-CRISPR-Cas13a technology.The method was evaluated by plasmids and clinical sample strains,and the detection was also performed by Quantitative real-time PCR(qPCR)method to compare the detection rate and consistency of the two methods.Results The RAA-CRISPR-Cas13a method can detect KPC plasmids and samples with a sensitivity of 1 copy/μl,which is higher than that of qPCR(101 copies/μl).Among the 30 clinical strains(including 25 CRKP strains and 5 CSKP strains),23 strains were detected to carry KPC gene by both RAA-CRISPR-Cas13a method and qPCR method,and 7 strains were not detected with KPC gene.The detection rate of KPC gene in the 25 CRKP strains was 92%(23/25).The positive coincidence rate of the two methods was 100%(23/23).Conclusions This study combined RAA amplification technology with CRISPR-Cas13a technology to establish a rapid and accurate method for detecting KPC carbapenemase gene.The method is useful for accurate screening of KPC carbapenemase-producing strains.It has a wide application prospect in drug resistance monitoring and infection control.

关 键 词：克雷伯菌 肺炎 微生物敏感性试验 分子检测 

分 类 号：R446.5[医药卫生—诊断学]