基于CRISPR/Cas13a系统建立大口黑鲈弹状病毒检测方法

A METHOD FOR DETECTION OF MICROPTERUS SALMOIDES RHABDOVIRUS BASED ON CRISPR/CAS13A SYSTEM

为了建立一种灵敏度高、特异性强并且适合现场诊断的大口黑鲈弹状病毒(Micropterus salmoides rhabdovirus,MSRV)检测方法,研究基于CRISPR-Cas13a系统并结合多酶恒温核酸快速扩增(MIRA)技术建立了一种MSRV的检测方法。实验对MSRV序列进行多重序列比对分析后,针对MSRV衣壳蛋白(CP)基因的特异性区域设计两个靶点,并通过体外转录成特异性的crRNA,同时设计合成MIRA引物序列实现目标序列的等温扩增,最后结合Cas13a蛋白、crRNA、恒温扩增体系构建检测体系,并从高效crRNA的选择、反应温度、ssRNA报告探针浓度和Cas13a与crRNA的浓度比四个方面优化了反应体系,并采用最优检测体系对大口黑鲈样本进行检测验证。结果显示,在20μL检测体系中加入200 nmol/L Cas13a、100 nmol/L crRNA1、100 nmol/L crRNA2及500 nmol/L ssRNA报告探针,在37℃的情况下能够获得最佳的检测效果。并且该检测体系可以检测到102 fM的MSRV病毒,具有良好的特异性和灵敏性,此外检测结果可直接通过紫外灯照射直接观察。研究基于CRISPR/Cas13a系统结合等温扩增MIRA技术建立的MSRV检测方法,可在室温下且不需要昂贵的仪器进行MSRV病毒检测,检测结果可以肉眼直接观察,能在多种场合下使用,研究结果为检测MSRV提供了有效方法。

The Micropterus salmoides rhabdovirus(MSRV)is a pathogenic RNA virus.Currently,detection methods for MSRV are limited and inconvenient.In order to establish a MSRV detection method with high sensitivity and specificity and suitable for on-site diagnosis,we developed an MSRV detection method based on the CRISPR-Cas13a system combined with the Multi Enzyme Thermostable Rapid Amplification of Nucleic Acids(MIRA)technique.After multiple sequence alignment analysis of MSRV sequences,two targets were designed for the specific region of MSRV capsid protein(CP)gene and transcribed into specific crRNA in vitro,and the MIRA primer sequence was designed and synthesized to achieve isothermal amplification of the target sequence.The reaction system was optimized in terms of the selection of efficient crRNA,reaction temperature,concentration of ssRNA reporter probe and the concentration ratio of Cas13a to crRNA,and the optimal detection system was validated for largemouth bass samples.The results showed that the addition of 200 nmol/L Cas13a,100 nmol/L crRNA1,100 nmol/L crRNA2 and 500 nmol/L ssRNA reporter probes in a 20μL assay system at 37℃gave the best detection results.The assay system demonstrated its capability to detect 102 fM MSRV virus with remarkable specificity and sensitivity.Moreover,the results could be directly observed by UV light irradiation.The MSRV assay established in this study is based on the integration of CRISPR/Cas13a system and the isothermal amplification MIRA technique.One of its significant advantages is its ability to detect MSRV virus at room temperature without the need for expensive instruments.Furthermore,the results can be directly observed by the naked eye and can be used in a variety of settings.Overall,the results of the study provide an effective method for detecting MSRV.