(H-287) Development of a rapid assay and a portable device to detect tuna species

Introduction:: Illegal, unreported, and unregulated (IUU) fishing violates the rights of Indigenous Peoples to traditional fishing grounds, compromises the food security of legitimate fishers and coastal populations, and facilitates human labor trafficking. Bluefin and yellowfin tuna, because of their highly valued meat, are among the 13 species most impacted by IUU [1]. Often their tails, fins, and heads are cut off before storage and transportation, making identification by phenotype nearly impossible. Thus, to accurately identify these tuna species, samples are traditionally shipped to off-site laboratories where an expensive polymerase chain reaction (PCR) instrument is used to detect species-specific DNA biomarkers. This process can take up to a week or more to receive results, preventing authorities from promptly acting on the information. To address this issue, we are developing a novel biological assay and a portable heater/reader device to detect different tuna species. Here we demonstrate a proof-of-concept workflow that can sensitively and specifically detect Yellowfin tuna (Thunnus albacares) samples within 30 min from samples to results.

Materials and Methods:: Our biological assay consists of two simple steps: 10-min DNA extraction by alkaline lysis (Fig. 1a) and a 20-min isothermal assay (Fig. 1b) using single-step tuna DNA amplification by Recombinase Polymerase Amplification (RPA) and tuna DNA detection by fluorescence using Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas12a). Our in-house lysis method was tested on commercial yellowfin tuna. DNA amplification yields from the lysate were compared to those from the DNA prepared by the Qiagen blood and tissue extraction kit. To design the RPA assay, we built on validated PCR primers [2] with modifications of 5’ sequences to meet RPA requirements. Protospacer adjacent motif (PAM)-free guide RNA for CRISPR-Cas12a designed to have opposite directions of RPA primers [3] were screened in a high-throughput format using a commercial qPCR machine. Additionally, a portable heating/detector device was built to simultaneously operate four samples. We used an ESP32 microcontroller and thermistor to control and monitor the temperature of the sample, which is heated by Positive Temperature Coefficient heating elements controlled by our Proportional-Integral-Derivative algorithm. 470nm LEDs were used to create fluorescence excitation and a 550 nm acrylic filter to isolate the fluorescence emission. For image analysis, we used the Python-based cv2 package, implementing a threshold method to eliminate samples with lower intensity than the positive threshold. To exclude bubbles or other artifacts, we used the contour method to circle all the objects in the image and applied the size threshold; objects with size >50th percentile are classified as positive.

Results, Conclusions, and Discussions:: Our newly-developed RPA successfully amplified yellowfin tuna DNA in lysates prepared by our in-house lysis method. The amplification yields of the DNA from lysates were comparable to those obtained from purified DNA from a Qiagen kit. We then used a commercial qPCR machine to characterize our single-tube RPA/CRISPR-Cas12a reactions (Fig. 1c). The final optimal assay specifically detected yellowfin tuna lysate with a detection time as early as 5 minutes and after 13-14 min crossed 1000 relatively reference units, a level detectable by our heater/detector device. Next, a panel of samples (i.e., water, bluefin tuna lysate, and yellowfin tuna lysate) was tested by our heater/detector device (Fig. 1d). Cell phone images (Fig. 1e) after 15 minutes of incubation were classified by our in-house software, correctly indicating positive results for yellowfin tuna lysates and negative results for bluefin lysates and water controls. These results demonstrate a successful proof-of-concept workflow for yellowfin tuna detection. Our next steps include the development of dried reagents of RPA/CRISPR-Cas12a to enable easy set-up and on-site detection in low-resource settings. Moreover, in development and testing, the computer monitored temperature and heater performance, while LEDs were controlled separately and connected using alligator clips. Next, we will integrate the lighting system with the microcontroller and embed everything within our portable device. In summary, the combination of our assay and portable device has the potential to offer a rapid method for identifying yellowfin tuna species on-site, which can aid the prevention of IUU fishing.

Acknowledgements (Optional): : We thank Dr. Eric Klavins and Dionne K. Vafeados for providing access to the BioFAB laboratory space and equipment. We thank Mike Roller for his advice on device development and our collaborators Jason Hoffman and Derek Zhu for developing a cell phone application for our next-generation device. We thank the pilot seed fund from the University of Washington Center for Environmental Forensic Science (Bohringer, Panpradist) and the University of Washington ITHS Early Investigator Catalyst Award (Panpradist) for their support on reagents and supplies used in this study. We also thank Mary Gates Research Scholarships (Kim and Ruslim). The funders have no roles in the study design or interpretation of results.

References (Optional): : [1] U.S. Department of State Office of Marine Conservation. Illegal, Unreported, and Unregulated Fishing. Accessed on April 30th, 2023 <https://www.state.gov/key-topics-office-of-marine-conservation/illegal-unreported-and-unregulated-fishing/>



[2] Lee GY, Suh SM, Lee YM, Kim HY. Multiplex PCR Assay for Simultaneous Identification of Five Types of Tuna (Katsuwonus pelamis, Thunnus alalonga, T. albacares, T. obesus and T. thynnus). Foods. 2022 Jan 20;11(3):280. doi: 10.3390/foods11030280. PMID: 35159432; PMCID: PMC8834044.



[3] Ding X, Yin K, Li Z, Lalla RV, Ballesteros E, Sfeir MM, Liu C. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020 Sep 18;11(1):4711. doi: 10.1038/s41467-020-18575-6. PMID: 32948757; PMCID: PMC7501862.