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

曹亚玲 ¹田原 ¹范子豪 ¹徐玲 ¹高耀 ¹张向颖 ¹任锋 ¹武昱

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作者信息

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
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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.

Abstract

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

Key words

Klebsiella pneumoniae/Microbial sensitivity tests/Molecular detection

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基金项目

国家自然科学基金(81770611)

国家自然科学基金(82002243)

北京自然科学基金-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)

首都卫生发展科研专项重点攻关项目(SF2021-1G-2181)

首都卫生发展科研专项重点攻关项目(SF2022-1-2182)

北京市临床诊疗技术示范应用与研究专项(Z191100006619096)

北京市临床诊疗技术示范应用与研究专项(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)

出版年

2024

中华检验医学杂志

中华医学会

中华检验医学杂志

CSTPCDCSCD北大核心

影响因子：1.402

ISSN：1009-9158

参考文献量13

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