Tilapia Lake Virus (TiLV) is a newly emerging, highly infectious pathogen in aquaculture. It has been reported to affect tilapia farming in 16 countries across multiple continents, with a cumulative mortality rate reaching up to 90 % in Nile tilapia (Oreochromis niloticus) (Surachetpong et al., 2020). Nile tilapia is one of the most important farmed fish species in Asia and Africa, with 6 million tonnes produced in 2020 (Lueangyangyuen et al., 2022). TiLV Disease (TiLVD) can lead to mortality rates as high as 90 %, resulting in significant losses for tilapia farms (Thawornwattana et al., 2020). Early detection of this virus plays a crucial role in facilitating prompt treatment, effectively reducing economic losses in the aquaculture (Kembou-Ringert et al., 2023).

Traditional methods for detecting TiLV rely on nucleic acid detection using polymerase chain reaction (PCR) techniques, including conventional simplex and multiplex PCR (Nicholson et al., 2018, Mugimba et al., 2018), real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) (Chengula et al., 2022), and loop-mediated isothermal amplification (LAMP) (Kampeera et al., 2021) assays. These methods require precise instrumentation and are prone to contamination, making them challenging to implement in clinical field testing. In recent years, some isothermal amplification techniques, such as recombinase polymerase amplification (RPA) (Wang et al., 2021), have been developed as attractive alternatives to traditional PCR methods due to their simplicity, speed, and low cost. However, RPA is susceptible to aerosol contamination leading to false-positive results (Liu et al., 2023), posing challenges in developing accurate and reliable point-of-care (POC) tests.

In recent years, research has found that CRISPR-Cas is an adaptive immune system in bacteria and archaea (Makarova et al., 2015). CRISPR, consists of a series of repetitive sequences with similarly sized unique spacers that record the nucleic acids of invading exogenous pathogens. When a pathogen invades again, the CRISPR sequences are transcribed and processed into small CRISPR RNA (crRNA, also known as guide RNA or gRNA). The crRNA guides Cas proteins to target and degrade the exogenous nucleic acids (Wu et al., 2021). Some of these Cas proteins, such as Cas12a, have been shown to activate their collateral cleavage activity upon target recognition, nonspecifically cleaving nearby single-stranded non-target nucleic acids (Chen et al., 2018). Utilizing this feature of Cas12a, researchers have developed a detection method for target genes by synthesizing single-stranded DNA (ssDNA) reporter molecules with luciferase and biotin modified at either end. When the target gene is present, the reporter molecule is cleaved, thereby enabling the detection of the target gene (Ma et al., 2022).As CRISPR-based nucleic acid detection technologies have been successively developed, researchers have combined the CRISPR/Cas system with isothermal amplification techniques to establish various nucleic acid detection platforms (Chen et al., 2023). For example, in 2021, the Optimized Rapid DNA Endonuclease Targeted CRISPR Trans Reporter (OR-DETECTR) based lateral flow assay was used for Corona Virus Disease 2019 (COVID-19) detection with a sensitivity of 2.5 copies per microliter, allowing for rapid and specific detection of the COVID-19 target at room temperature (Sun et al., 2021). In 2019, the research group led by Liang Mindong developed a simple, ultra-sensitive, rapid, and high-throughput platform based on CRISPR/Cas12a for detecting small molecules, successfully detecting uric acid in human blood samples with sensitivity at the nanomolar level (Liang et al., 2019).

CRISPR-Cas12a is an engineered nucleic acid enzyme belonging to the CRISPR system (Kleinstiver et al., 2019, Turner et al., 2023, Widziolek et al., 2021), capable of triggering indiscriminate non-specific single-strand DNA cleavage upon recognizing specific DNA sequences (Swarts and Jinek, 2019, Zhang et al., 2023). When combined with RT-RAA for virus detection, RT-RAA first rapidly and efficiently amplifies the virus's RNA into cDNA at room temperature(Jedrzejczyk et al., 2022; Uno et al., 2023). Then, by designing specific crRNA, CRISPR-Cas12a can specifically recognize and bind to the amplified target DNA sequence (Kwak et al., 2023, Park et al., 2021). Once recognized, Cas12a activates its cleavage activity (Liu et al., 2021), cutting the reporter molecule linked to the viral DNA, thereby achieving signal amplification and visualization (Dai et al., 2019, Liu et al., 2022).This combination technique offers significant sensitivity and specificity, enabling accurate detection even at very low viral loads (Zhao et al., 2021), which is crucial for early diagnosis and disease prevention and control (Jiang et al., 2023). Moreover, this method does not require expensive laboratory equipment and highly trained personnel, significantly reducing detection costs and operational complexity (Hu et al., 2022), making it particularly suitable for resource-limited areas.

By combining RT-RAA and CRISPR-Cas12a technologies, this study not only provides an innovative method for rapid detection of TiLV but also has the potential to greatly improve disease management and control strategies, reducing the economic and social impacts of TiLV outbreaks. This holds significant positive implications for the global tilapia farming industry and related aquaculture sectors. Sukonta et al. (2022) first applied RT-RPA-CRISPR/Cas12a to detect TiLV. However, its detection limit of 200 copies per reaction is still suboptimal. Therefore, improving this technology for TiLV diagnostics is necessary and worthwhile, given CRISPR’s advantages and field applications.