A novel dual CRISPR-Cas assay for detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp without false positives from its endogenous viral elements (EVEs)

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Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus posing significant economic challenges in shrimp aquaculture. Depending on the shrimp species, IHHNV infections can lead to mass mortalities, deformities, or growth retardation, all of which impact production output. Timely identification of IHHNV, particularly using nucleic acid detection methods, is paramount for effective prevention. However, PCR and methods relying on nucleic acid probes face challenges due to the presence of chromosome-integrated, endogenous viral elements (EVEs) of IHHNV sharing identical sequences to their infectious counterparts. While long-range PCR assays, such as LongAmp PCR (LA-PCR), have shown promise in minimizing false positive results, their applicability in the field is limited by their long turnaround times and reliance on costly equipment. To address these limitations, we have developed a rapid and potentially field-deployable platform termed Cas9-RPA-Cas12a (“CRPAC”) capable of differentiating the infectious form of IHHNV from EVEs, capitalizing on the fact that the genome of IHHNV is single-stranded, while EVEs are double-stranded DNA. This method employs Cas9 endonuclease to deplete EVEs, thereby enriching for single-stranded genomic DNA. This enriched DNA is then subjected to recombinase polymerase amplification (RPA) coupled with the Cas12a diagnostic assay. Here, we show that CRPAC gives positive results exclusively for infectious IHHNV, appearing as strong fluorescence or a distinct pattern on lateral flow strips that facilitates naked-eye evaluation. In field sample testing, CRPAC demonstrated excellent agreement with LA-PCR, identifying 8 out of 16 samples containing infectious IHHNV, while the WOAH-recommended PCR assays gave false positive results to all samples containing EVEs. Collectively, our findings underscore the utility of CRPAC as a robust and field-adaptable tool for distinguishing between infectious IHHNV and EVEs, irrespective of their nucleotide sequence similarities.