Abstract
The red-spotted grouper nervous necrosis virus (RGNNV) is the most widely distributed nervous necrosis virus (NNV) and causes a significant risk to aquatic food safety. Though a few CRISPR-based aquatic pathogen detection methods have been reported recently, the absence of the PAM site of RGNNV specific gene RNA2 impedes its CRISPR-based diagnostic. Herein, we developed a rapid and accurate homemade isothermal amplification-initiated Cas14a assay (HiaCas14a) for RGNNV detection. The introduction of the PAM site into RT-LAMP specific amplicons of RGNNV RNA2 unlocked its PAM restriction. Furthermore, fish genomic RNA purified by magnetic beads and polymerases used in RT-LAMP prepared by ourselves obviously gave the system an enhanced convenience and a reduced cost. HiaCas14a detected 63.4 aM RNA with excellent specificity and sensitivity quantitatively, and it demonstrated a high accuracy of field sample validation results compared with RT-qPCR. The method provided an accurate, low-cost, and sensitive tool for aquatic pathogen detection in resource-poor areas.
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