Aquatic animal disease management is crucial to aquaculture, and thus, investment in disease control is vital to success and sustainability. Intensification of shrimp farming has led to the development of several diseases which result in enormous loss in shrimp production within the farm with subsequent significant economic impact to the farmer. The current diagnostic methods used by local farms and regulatory bodies such as Singapore Food Agency (SFA) is real-time polymerase chain reaction (PCR). The real-time PCR is lab based, requires expensive equipment and skilled personnel for data interpretation. Therefore, there is a pressing need to develop a diagnostic tool that is not only sensitive and specific but also simple to execute and capable of providing rapid results either onsite or directly at shrimp farms. In response to this need, we have successfully leveraged Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a technologies to devise a novel diagnostic test. This test enables the simultaneous detection of three major pathogens affecting shrimp: white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Enterocytozoon hepatopenaei (EHP).
Initially, we conducted monoplex RPA assays followed by detection with Cas12a, achieving robust fluorescence signals for all three pathogens. Encouraged by these results, we optimized multiplex RPA (mRPA) assays for simultaneous detection of the three pathogens. The mRPA products, labeled with distinct fluorophores, exhibited intense fluorescence corresponding to WSSV, IHHNV, and EHP. Subsequent limit of detection (LOD) studies revealed a sensitivity of 1 copy per reaction for monoplex RPA/Cas12a assays and 50 copies per reaction for multiplex RPA/Cas12a assays. Furthermore, validation using simulated field samples demonstrated the reproducibility and accuracy of our test in detecting pathogens within the complex genomic background of shrimp.
In conclusion, our CROP test kit offers a rapid, sensitive, and multiplexed approach for diagnosing shrimp diseases. It holds immense promise for improving disease control practices, minimizing economic losses, and fostering the long-term sustainability of the industry. Moving forward, we are working on integrating these technologies into a microfluidic device to facilitate on-farm testing, thereby reducing the need for specialized equipment and expertise. This advancement holds promise for enhancing disease management practices and ensuring the sustainability of shrimp aquaculture.