One-Pot RAA-CRISPR/Cas12a Assay for Rapid Detection of Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV) in Shrimp Aquaculture.

Abstract

The infectious hypodermal and haematopoietic necrosis virus (IHHNV) represents a significant viral threat to global shrimp aquaculture, leading to considerable economic losses. In this study, we have developed a one-step, one-pot isothermal assay for the detection of IHHNV, employing recombinase-aided amplification in conjunction with clustered regularly interspaced short palindromic repeats-Cas12a (RAA-CRISPR/Cas12a). The assay is performed at a constant temperature of 37°C, achieving a detection limit of 10 copies per reaction for the fluorescence assay and 1 copy per reaction for the lateral flow dipstick (LFD) assay within a 60-min timeframe. Additionally, we evaluated the assay against four other prevalent shrimp pathogens (WSSV, DIV1, EHP, VpAHPND) and observed no cross-reactivity. This straightforward detection method exhibits high sensitivity and specificity for IHHNV, offering a promising approach for early and rapid field diagnosis.