Thawatchai Chaijarasphong

Streptococcus agalactiae is a primary pathogen responsible for significant mortality in tilapia, a critical and affordable source of animal protein. Among the various serotypes affecting aquaculture, serotype III is increasingly recognized for its high virulence and zoonotic potential, with multidrug resistance reported in some lineages. However, diagnostic tools that combine serotype‐level resolution with field‐deployability remain scarce. In this study, we developed a rapid and specific CRISPR‐based detection platform targeting the cpsH gene of S. agalactiae serotype III. Combining recombinase polymerase amplification and CRISPR detection, the standard two‐step RPA‐Cas12a assay achieved a limit of detection (LoD) of 100 copies per reaction, with no cross‐reactivity observed against closely related cpsH homologues from serotypes Ia, Ib and VI. The entire workflow can be completed within 1 h at a constant temperature of 37°C, providing visual readouts that can be easily and unambiguously interpreted. Furthermore, we demonstrated that the assay can be streamlined into a photocontrolled one‐pot format, which reduces the risk of aerosol contamination while maintaining the same sensitivity as the two‐step protocol. When evaluated against simulated DNA‐spiked samples and field samples, both assay formats exhibited 100% concordance with each other and with the conventional PCR method, highlighting the potential of this platform as a step toward rapid, serotype‐specific surveillance in aquaculture.

White spot syndrome virus (WSSV) and Enterocytozoon hepatopenaei (EHP) represent the most economically destructive pathogens in the current shrimp industry. WSSV causes white spot disease (WSD) responsible for rapid shrimp mortality, while EHP stunts growth and therefore reduces overall productivity. Despite the importance of timely disease detection, current diagnostic methods for WSSV and EHP are typically singleplex, and those offering multiplex detection face issues such as complexity, low field compatibility and/or low sensitivity. Here, we introduce an orthogonal, multiplex CRISPR‐Cas assay for concomitant detection of WSSV and EHP. This method combines recombinase polymerase amplification (RPA) for target DNA enrichment with Cas12a and Cas13a enzymes for fluorescent detection. This assay produces distinct fluorescent colours for different diagnostic outcomes, allowing naked eye visualisation without ambiguity. Further validation reveals that the assay detects as few as 20 and 200 copies of target DNA from EHP and WSSV, respectively, while producing no false positives with DNA from other shrimp pathogens. Moreover, the assay excellently agrees with established PCR methods in evaluation of clinical samples. Requiring only 37°C and less than an hour to complete, multiplex CRISPR‐Cas assay presents a promising tool for onsite diagnostics, offering high accuracy while saving time and resources.

Tilapia is an affordable protein source farmed in over 140 countries with the majority of production in low‐ and middle‐income countries. Intensification of tilapia farming has exacerbated losses caused by emerging and re‐emerging infectious diseases. Disease diagnostics play a crucial role in biosecurity and health management to mitigate disease loss and improve animal welfare in aquaculture. Three continuous levels of diagnostics (I, II and III) for aquatic species have been proposed by Food and Agriculture Organization of the United Nations (FAO) and the Network of Aquaculture Centers in Asia and the Pacific (NACA) to promote the integration of basic and advanced methods to achieve accurate and meaningful interpretation of diagnostic results. However, the recent proliferation of cutting‐edge molecular methods applied in the diagnosis of diseases of aquacultured animals has shifted the focus of researchers and users away from basic approaches and toward molecular diagnostics, despite the fact that many diseases can be rapidly diagnosed using inexpensive, simple microscopic examination and that most emerging diseases in aquaculture were discovered by histopathology. This review, therefore, revisits and highlights the importance of the three levels of diagnostics for diseases of tilapia, particularly the frequently overlooked basic procedures (e.g., case history records, gross pathology, presumptive diagnostic methods and histopathology). The review also covers current and emerging molecular diagnostic technologies for tilapia pathogens including polymerase chain reaction methods (conventional, quantitative, digital), isothermal amplification methods Loop‐mediated Isothermal Amplification (LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR)‐based detection, lateral flow immunoassays, as well as discussing what is on the horizon for tilapia disease diagnostics (next generation sequencing, artificial intelligence, environmental Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) and point‐of‐care testing) providing a future vision for transferring these technologies to farmers and stakeholders for a sustainable aquatic food system transformation.

Acute Hepatopancreatic Necrosis Disease (AHPND), caused by bacterial isolates expressing PirAB binary toxins, represents the severest and most economically destructive disease affecting penaeid shrimp. Its rapid disease progression and associated massive mortalities call for vigilant monitoring and early diagnosis, but molecular detection methods that simultaneously satisfy the requirements of sensitivity, specificity, and portability are still scarce. In this work, the CRISPR‐Cas12a technology was harnessed for the development of two fluorescent assays compatible with naked‐eye visualization. The first assay, AP4‐Cas12a, was based on the OIE‐recommended AP4 two‐tubed nested PCR method and was designed to bypass the time‐consuming and potentially hazardous agarose gel electrophoresis step. Using AP4‐Cas12a, the detection limit of 10 copies per reaction could be achieved within less than 30 minutes post‐PCR. The second assay, RPA‐Cas12a, utilized recombinase polymerase amplification (RPA) to rapidly and isothermally amplify the target DNA, followed by amplicon detection by Cas12a, resulting in a protocol that can be completed in less than an hour at a constant temperature of 37°C. The detection limit of RPA‐Cas12a is 100 copies of plasmid DNA or 100 fg of bacterial genomic DNA per reaction. Importantly, we validated that both assays are compatible with a previously reported smartphone‐based device for facile visualization of fluorescence, thereby providing an affordable option that requires less consumables than lateral flow detection. Using this portable device for readouts, the AP4‐Cas12a and RPA‐Cas12a methods showed excellent concordance with the AP4‐agarose gel electrophoresis approach in the evaluation of clinical samples. Therefore, the developed Cas12a assays have the potential to streamline both in‐laboratory and onsite diagnosis of AHPND.

Scale drop disease virus (SDDV) is a major pathogen of Asian sea bass that has emerged in many countries across the Asia Pacific since 1992 and carries the potential to cause drastic economic losses to the aquaculture sector. The lack of an approved vaccine for SDDV necessitates timely prevention as the first line of defence against the disease, but current diagnostic platforms still face challenges that render them incompatible with field applications, particularly in resource‐limited settings. Here, we developed a novel detection platform for SDDV based on a CRISPR‐Cas12a‐based nucleic acid detection technology combined with recombinase polymerase amplification (RPA‐Cas12a). Using the viral adenosine triphosphatase (SDDV‐ATPase) gene as a target, we achieved the detection limit of 40 copies per reaction and high specificity for SDDV. The coupling with fluorescence and lateral flow readouts enables naked‐eye visualization and straightforward data interpretation requiring minimal scientific background. Compared with semi‐nested PCR in field sample evaluation, our RPA‐Cas12a assay is more sensitive and capable of detecting SDDV in asymptomatic fish. Importantly, the entire workflow can be carried out at a constant temperature of 37°C within an hour from start to finish, thus removing the need for an expensive thermal cycling apparatus and long turnaround times associated with PCR‐based methods. Therefore, owing to its high accuracy, rapidity and user‐friendliness, the developed RPA‐Cas12a platform shows the potential for diagnosis of SDDV at point of need and could be a valuable tool to help protect fish farming communities from large‐scale epidemics.