Highly sensitive detection of white spot syndrome virus with an RPA-CRISPR combined one-pot method

Abstract

The white spot syndrome virus (WSSV) is a pathogenic virus that has caused serious problems in aquaculture. An effective therapy for its infection is not yet available, and it is essential to develop a fast-response diagnosis method. This study established an RPA and CRISPR-Cas12a combined one-pot method for WSSV detection. The one-pot method was achieved by rational design and systematic optimization of the reaction, including concentrations of Cas12a and crRNA, and the buffer condition. The sensitivity reached 101 copies/reaction, which was in the highly sensitive range when compared to all the available molecular detection assays. Moreover, the specificity and field sample validation results were satisfactory. The method has integrated the potent amplification capacity of RPA and the precise cleavage-activation property of CRISPR-Cas12a into one simultaneous reaction, thus has enhanced convenience and less risk of carry-over contaminations. The method is conducted under 37 °C and only demands for minimum laboratorial support. It provides a convenient and reliable on-site diagnosis tool for WSSV infection in aquaculture.