基于CRISPR/Cas13a的马尔堡病毒核酸检测方法的建立

A CRISPR/Cas13a-based nucleic acid detection method for Marburg virus

摘要:

【目的】 建立一种基于成簇的规律间隔短回文重复序列及其相关蛋白13a (clustered regularly interspaced short palindromic repeats/associated protein 13a, CRISPR/Cas13a)，针对马尔堡病毒核酸的快速检测方法。【方法】 根据马尔堡病毒核蛋白(nucleoprotein, NP)基因保守区序列设计合成特异性的逆转录酶重组酶介导链替换核酸扩增(reverse transcription recombinase aided amplification, RT-RAA)引物及CRISPR RNA (crRNA)，利用RT-RAA等温扩增技术对靶序列进行扩增，通过CRISPR/Cas13a系统检测扩增产物，并结合消线法(easy-readout and sensitive enhanced, ERASE)侧流层析试纸进行结果判读。最后利用国家标准品对建立的新方法的灵敏度和特异性进行评估。【结果】 筛选出一组针对马尔堡病毒NP基因的高效扩增引物和crRNA，建立了用于马尔堡病毒检测的CRISPR-ERASE方法，可在1 h内检测出浓度为1 copy/μL目标核酸，并与其他多种病原体无交叉反应。【结论】 本研究基于CRISPR/Cas13a建立了一种快速、简便、高灵敏度和高特异性的马尔堡病毒核酸检测方法。

Abstract:

[Objective] To develop a rapid nucleic acid detection method for Marburg virus based on clustered regularly interspaced short palindromic repeats/associated protein 13a (CRISPR/Cas13a). [Methods] According to the conserved region of Marburg virus nucleoprotein (NP) gene, specific primers for reverse transcription recombinase-aided amplification (RT-RAA) and CRISPR RNA (crRNA) were designed and synthesized. RT-RAA was employed to amplify the target sequence. The amplification products were detected by the CRISPR-Cas13a system, and the results were interpreted by easy-readout and sensitive enhanced (ERASE) lateral flow test strips. Finally, the national reference panel was used to evaluate the sensitivity and specificity of the new method. [Results] A set of high-efficiency RT-RAA primers and crRNA targeting Marburg virus NP gene was screened, on the basis of which a CRISPR-ERASE method for the detection of Marburg virus was developed. The target nucleic acid with a concentration of 1 copy/μL could be detected within 1 h, and there was no cross-reaction with other several pathogens. [Conclusion] In this study, a rapid, simple, highly sensitive, and specific nucleic acid detection method for Marburg virus was developed based on CRISPR/Cas13a.