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

ABSTRACT

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 × 10 ¹ 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.