An enzymatic recombinase amplification assay combined with CRISPR-Cas12a for the rapid detection of acute hepatopancreatic necrosis disease in shrimp Penaeus vannamei

The shrimp farming sector has faced significant hurdles due to acute hepatopancreatic necrosis disease (AHPND). In this study, an attempt was made to combine the enzymatic recombinase amplification (ERA) assay and CRISPR-Cas12a (ERA-Cas12a) for the rapid detection of AHPND. A set of crRNA and ssDNA with high sensitivity and good specificity was screened, and the best optimized one-tube system was obtained. Our additional analysis focused on the sensitivity and specificity of ERA-Cas12a, contrasting it with the established methodology of the World Organization for Animal Health (WOAH). The ERA-Cas12a could deliver the desired outcomes in under 40 min with a detection limit of 2 x 10(3) copies/mu L and showed no interaction with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), Taura syndrome virus (TSV), covert mortality nodavirus (CMNV), and decapod iridescent virus 1 (DIV1). Our actual sample detection results showed that the ERA-Cas12a system has the advantage of being more intuitive and fast without losing too much sensitivity compared with the nested PCR and quantitative real-time PCR (qPCR). Moreover, the detection results could be directly observed under blue light irradiation. All these results indicate that ERA-Cas12a provides a rapid and specific diagnosis of AHPND, which could further reduce the dependence on the instrument and effectively reduce aerosol pollution. It could be helpful in the prevention of shrimp disease and in reducing the economic loss of shrimp aquaculture.