Field-Deployable RPA-CRISPR/Cas12a Detection of Decapod Iridescent Virus 1 in Shrimp

Decapod iridovirus 1 (DIV1) is a highly lethal pathogen that infects decapod crustaceans including Litopenaeus vannamei, causing mass mortality in cultured shrimp and severe economic losses worldwide. The ATPase gene is a highly conserved region within the DIV1 genome, plays a critical role in viral replication and represents an ideal target for molecular diagnostic development. In this study, we established a rapid, sensitive and field-adaptable detection platform for DIV1 by integrating recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system. RPA enables efficient isothermal amplification of target nucleic acids, achieving exponential enrichment of the target nucleic acids and exerting the function of signal amplification. While the CRISPR/Cas12a system upon crRNA-guided specific recognition of the amplicon, triggers robust trans-cleavage activity against reporter probes for signal generation and readout. After systematic optimization, the RPA reaction was performed at 38°C for 10 min and the CRISPR-Cas12a reaction was conducted at 37°C for 20 min. The integrated two-step workflow completed detection within 40 min, with a limit of detection of 2.3 × 10¹ copies/μL. Specificity evaluation confirmed that the RPA-CRISPR/Cas12a assay exclusively recognised DIV1 without cross-reaction with other major shrimp pathogens. Further validation using clinical shrimp samples demonstrated stable and reliable performance, supporting its practical utility in aquaculture settings. In conclusion, the established CRISPR/Cas12a-based detection platform provides a robust technical tool for early warning and on-site rapid screening of DIV1, facilitating timely disease control and risk management in shrimp farming.