Development of a Single-Tube Asymmetric ERA-CRISPR/Cas12a Assay for Rapid Visual Detection of Enterocytozoon hepatopenaei in Shrimp

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) is a major pathogen causing severe growth retardation in shrimp, leading to substantial economic losses in global aquaculture. To address the urgent need for accurate, rapid, and field-deployable diagnostic tools for EHP, this study developed a novel one-pot detection platform by integrating asymmetric Enzymatic Recombinase Amplification (aERA) with a PAM-independent CRISPR/Cas12a system (AYERA-Cas12a) based on ssDNA activation. This design circumvents the compatibility challenge between isothermal amplification and CRISPR activity in a single tube by generating single-stranded DNA amplicons that activate Cas12a without requiring a PAM sequence. The assay operates at a constant temperature of 46 °C and completes detection within 15 min. It achieves a sensitivity of 10 copies/μL, equivalent to qPCR, and shows no cross-reactivity with six other prevalent shrimp pathogens. Validation using 56 clinical shrimp (Litopenaeus vannamei, L. vannamei) samples demonstrated complete agreement with qPCR results. With its simple procedure, isothermal conditions, and clear endpoint fluorescence readout under blue light, the AYERA-Cas12a platform is suitable for point-of-care testing (POCT). This work provides a user-friendly tool for the on-site surveillance and early diagnosis of EHP, offering significant potential for improving disease management in shrimp farming.