High-efficient molecular detection system termed RAA-based CRISPR-Cas13a for novel duck orthoreovirus

The novel duck orthoreovirus (NDRV) is an immunosuppressive pathogen that significantly impacts the health of waterfowl breeding. Accurate, efficient, and convenient detection techniques are crucial for the prevention and control of NDRV, particularly in terms of field detection. By employing recombinase aided amplification (RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we have developed a highly sensitive enzymatic molecular system that combines Cas13a with T7 in vitro transcription and RAA, enabling efficient and accurate detection of NDRV at a sensitivity level of 10° copies/μL. Furthermore, the integration of portable lateral flow dipstick can effectively reduce the point-of-care testing time to 40 min, while exhibiting no cross-reactivity with duck hepatitis a virus, Tembusu virus and novel duck-origin goose parvovirus. Both this system and the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR) method demonstrated a consistent 100% accuracy in clinical samples. This study facilitated the development of an optimized assay, which enables specific detection of NDRV through a simplified procedure and significantly reduces the risk of contamination. This highlights the potential applicability of this assay for point-of-care testing.