Establishment and evaluation of a RAA-CRISPR-Cas13a method for detecting KPC carbapenemase genes
10.3760/cma.j.cn114452-20231009-00195
- VernacularTitle:基于RAA-CRISPR-Cas13a检测KPC型碳青霉烯酶基因方法的建立及评价
- Author:
Yaling CAO
1
;
Yuan TIAN
;
Zihao FAN
;
Ling XU
;
Yao GAO
;
Xiangying ZHANG
;
Feng REN
Author Information
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
- Keywords:
Klebsiella pneumoniae;
Microbial sensitivity tests;
Molecular detection
- From:
Chinese Journal of Laboratory Medicine
2024;47(2):159-164
- CountryChina
- Language:Chinese
-
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 (10 1 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.
