基于CRISPR/Cas13a的金黄色葡萄球菌mecA耐药基因检测方法的建立

A CRISPR/Cas13a-based detection method for the drug resistance gene mecA of Staphylococcus aureus

摘要:

【目的】 针对目前耐药基因检测通常需要依赖专业检测设施和设备,仍缺乏耐药基因快速检测方法这一问题,旨在建立一种基于成簇规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)的金黄色葡萄球菌(Staphylococcus aureus) mecA耐药基因快速检测方法。【方法】 首先在mecA基因序列的保守区中设计筛选出灵敏度较高的重组酶介导链替换核酸扩增(recombinase aided amplification, RAA)引物和CRISPR RNA (CRISPR RNA, crRNA),通过结合消线法核酸检测试纸技术(easy-readout and sensitive enhanced, ERASE)建立针对mecA基因的检测方法,最后利用模拟样本及临床分离样本对建立的新方法与传统方法进行比较。【结果】 成功筛选出了1组针对mecA耐药基因的高效扩增引物和crRNA,并建立了基于CRISPR-ERASE的mecA耐药基因高灵敏核酸检测方法,最低检出限为10 copies/μL,在32株临床分离的金黄色葡萄球菌中,该方法共检出24株mecA耐药基因阳性菌株,与药敏试验及荧光定量PCR (quantitative real-time PCR, qPCR)检测结果符合率为100%。【结论】 建立了一种基于CRISPR-ERASE核酸检测试纸技术的简单、高灵敏的mecA耐药基因检测方法。

关键词:CRISPR;等温扩增;mecA;核酸检测;ERASE试纸

Abstract:

[Objective] To address the problem that the detection of drug resistance gene usually relies on specialized devices and the lack of rapid detection methods, we aimed to establish a rapid detection method for the drug resistance gene mecA of Staphylococcus aureus based on clustered regularly interspaced short palindromic repeats (CRISPR). [Methods] Firstly, we designed and screened the recombinase-aided amplification (RAA) primers and CRISPR RNA (crRNA) with high sensitivity according to the conserved region of mecA. Then, we established the detection method for mecA with the easy-readout and sensitive enhanced (ERASE) strip. Finally, we used simulated samples and clinical isolated samples to compare the established method with the traditional method. [Results] A set of efficient RAA primers and crRNAs targeting mecA were successfully screened out, and a highly sensitive CRISPR-ERASE-based nucleic acid detection method was established for mecA. This method can detect mecA at a minimum concentration of 10 copies/μL. Among 32 clinical isolates of S. aureus, 24 strains were tested positive for mecA by the established method, which was in 100% agreement with the results of antimicrobial susceptibility test and quantitative real-time PCR (qPCR). [Conclusion] A CRISPR-ERASE-based simple and sensitive method for detecting the drug resistance gene mecA was established.

Key words:CRISPR;isothermal amplification;mecA;nucleic acid detection;ERASE strips