Rapid and facile detection of largemouth bass ranavirus with CRISPR/Cas13a.

Largemouth bass ranavirus (LMBV) is an epidemic disease that seriously jeopardizes the culture of largemouth bass（Micropterus salmoides）, and it has a very high incidence in largemouth bass. Once an outbreak occurs, it may directly lead to the failure of the culture, resulting in substantial economic losses, but there is no effective vaccine or special effective drug yet. Consequently, it is important to establish an accurate, sensitive, convenient and specific detection approach for preventing LMBV infection. The recombinant enzyme-assisted amplification (RAA) technology was used in combination with clustered regularly interspaced short palindromic repeats (CRISPR), and associated protein 13a (CRISPR/Cas13a) to detect LMBV. We designed RAA primers and CRISPR RNA (crRNA) that targeted the conserved region in the LMBV main capsid protein (MCP) gene, amplified sample nucleic acids using the RAA technology, performed CRISPR/Cas13a fluorescence detection and evaluated the sensitivity and specificity of the established method with qPCR as a control method. This technique was able to determine the results by collecting fluorescence signals, visualizing fluorescence by UV excitation and combining with lateral flow strips (LFS). The sensitivity and specificity of the established method were consistent with the qPCR method. Besides, it was performed at a constant temperature of 37 °C and the sensitivity of the reaction system was 3.1 × 10

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Declaration of competing interest The authors state that they have no known economic interests or personal relationships that may affect the work reported in this paper.