Dual-CRISPR/Cas12a-assisted RT-RAA visualization system for rapid on-site detection of nervous necrosis virus (NNV)

Abstract

Background

Nervous necrosis virus (NNV) poses a severe threat to the aquaculture industry, particularly infecting fish fry with devastating mortality rates and inflicting heavy economic losses. Traditional detection methods, such as cell culture and conventional RT-PCR, are not only time-consuming and require specialized laboratory facilities but also hard to eliminate contamination. Rapid and accurate on-site detection methods in aquaculture settings are crucial for effective control of NNV outbreaks in fish farms.

Results

This study developed a one-tube visualization system for rapid and precise identification of NNV in a pond-side setting. This system utilizes the dual-clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-assisted reverse transcription-recombinase aided amplification (RT-RAA) detection method, employing fluorescence intensity to indicate positive results for easy interpretation by field operators. The key to this system involved the meticulous selection of RT-RAA primer sets and CRISPR RNA (crRNA) primer sets targeting two genes of NNV, the capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), distributing on two particles of genomic sequences. The assay demonstrated a speed and efficiency process within 30 min and a detection limit of 0.5 copies/μL, achieving 100% accuracy when compared to qRT-PCR. The practical utility and effectiveness were validated by using 32 field samples. The results underscored the simplicity, rapidity, and reliability of the system, confirming its potential as a robust tool for NNV diagnosis in fish farms.

Significance

This study introduces the first application of a dual-CRISPR/Cas12a-assisted RT-RAA visualization system for diagnosing NNV infections. The novel approach substantially enhances on-site diagnostic capabilities, offering a rapid, reliable, and cost-effective solution for fish farm operators. This innovation not only streamlines the detection process but also ensures timely intervention, thereby mitigating the impact of NNV on aquaculture.