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

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 20 μL 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.