Development of efficient, sensitive, and specific detection method for Encephalomyocarditis virus based on CRISPR/Cas13a

Abstract

The Encephalomyocarditis virus (EMCV) is one of the major zoonosis pathogens, and it can cause acute myocarditis in young pigs or reproductive failure in sows. EMCV has been recognized as a pathogen infecting many species and causes substantial economic losses worldwide. Therefore, the development of a rapid, sensitive, and accurate detection of this virus is essential for the diagnosis and control of the EMCV-induced disease. The RNA-guiding, RNA-targeting CRISPR effector CRISPR/Cas13a (Cas13a, previously known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon the target recognition. When the crRNA of LwCas13a binds to the target RNA, the collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA. In this study, we developed an efficient, sensitive, and specific EMCV detection method based on the collateral cleavage activity of LwCas13a by combining recombinase-aided amplification (RAA) and a lateral flow strip. This method was an isothermal detection at 37 °C, which allowed visual observation by the naked eyes. We also optimized the reaction conditions of this method, and the detection result could be obtained within 60 min. The sensitivity of our method reached up to 10¹ copies/µL. Furthermore, no cross-reactions with other 8 major swine viruses were observed, indicating the excellent specificity of this method. At the same time, the assay had a 100 % coincidence rate with qPCR detection of the EMCV in 37 clinical samples. In addition, our developed method requires only 2-step operations and basic equipment, and thus it is simple and inexpensive. Overall, CRISPR/Cas13a-based detection has a great application potential for the EMCV detection.

Keywords: CRISPR/Cas13a; Detection method; Encephalomyocarditis virus; Lateral flow strip; RAA.