Multiplex CRISPR-Cas Assay for Rapid, Isothermal and Visual Detection of White Spot Syndrome Virus (WSSV) and Enterocytozoon hepatopenaei (EHP) in Penaeid Shrimp

White spot syndrome virus (WSSV) and Enterocytozoon hepatopenaei (EHP) represent the most economically destructive pathogens in the current shrimp industry. WSSV causes white spot disease (WSD) responsible for rapid shrimp mortality, while EHP stunts growth and therefore reduces overall productivity. Despite the importance of timely disease detection, current diagnostic methods for WSSV and EHP are typically singleplex, and those offering multiplex detection face issues such as complexity, low field compatibility and/or low sensitivity. Here, we introduce an orthogonal, multiplex CRISPR-Cas assay for concomitant detection of WSSV and EHP. This method combines recombinase polymerase amplification (RPA) for target DNA enrichment with Cas12a and Cas13a enzymes for fluorescent detection. This assay produces distinct fluorescent colours for different diagnostic outcomes, allowing naked eye visualisation without ambiguity. Further validation reveals that the assay detects as few as 20 and 200 copies of target DNA from EHP and WSSV, respectively, while producing no false positives with DNA from other shrimp pathogens. Moreover, the assay excellently agrees with established PCR methods in evaluation of clinical samples. Requiring only 37 degrees C and less than an hour to complete, multiplex CRISPR-Cas assay presents a promising tool for onsite diagnostics, offering high accuracy while saving time and resources.