(H-292) Fish Oligonucleotide Ligation Assay: a molecular classifier assay for rapid, portable, and inexpensive Pacific cod (Gadus macrocephalus) population assignment

Introduction:: Fish and seafood play a critical role in human food security and nutrition. To achieve sustainable fishing, we need accessible technologies to detect illegal, unreported, and unregulated fishing. DNA-based technologies allow the detection of genetic biomarkers such as single nucleotide polymorphisms (SNPs) in the DNA of a specific species, population, or region of origin. However, despite rapid advances in sequencing technologies, most SNP assays can take days or weeks to complete. Complexities limit their use in low-resource settings as they require well-equipped facilities and trained personnel. To address this need, we have developed a rapid DNA testing platform for species and source-population identification. We first focused on a commercially important species, the Pacific cod (Gadus macrocephalus)¹, which shows high population differentiation at two adjacent SNPs within 20 base pairs of the zona pellucida 3 (ZP3)². Here we developed and evaluated a molecular classifier assay, "Fish-OLA" (Fish Oligonucleotide Ligation Assay), to distinguish Pacific cod from the Bering Sea and Aleutian Island (BSAI) versus those from other stocks.


Materials and Methods:: Fish-OLA workflow includes four main steps (Fig. 1a): sample preparation (10 - 20 min), PCR amplification (60 min), ligation (20 min), and lateral flow detection (20 min). The Fish-OLA kit contains lyophilized reagents to facilitate assay setup and lateral flow tests to enable visual readout. Purified cod DNA using the Boom method or crude lysates prepared by the alkaline lysis method was PCR amplified using newly designed primers. Amplicons then underwent ligation in the presence of three allele-specific probes spanning the two adjacent signature SNPs. Only when the two biomarker SNPs are present will three probes be ligated.

Additionally, two additional control probes targeting a conserved region upstream of the two biomarker SNPs were present to serve as the ligation control. The resultant ligation mix was then visualized using lateral flow strips. Cod from the BSAI stock would be expected to generate visible bands on the BSAI "B," ligation control "C," and Flow Control "F" lines (Fig. 1b and 1c). Conversely, other cod stocks expectedly generate the "C" and "F" lines. To assess performance on the classification of cod origins, Fish-OLA kits were evaluated on blinded panels of extracted cod DNA (n=200) and crude lysates directly from ethanol-preserved cod fin clips (n=35). These samples were previously collected from the Eastern Bering Sea shelf (n=47 from Pervenets Canyon; n=53 from Zhemchug Canyon), the southern Gulf of Alaska (n=80), and Puget Sound (n=20). Lastly, we investigated the feasibility of the Fish-OLA kits on first-time users (48 users); each processed two samples.


Results, Conclusions, and Discussions:: Blind evaluation of Fish-OLA revealed accurate identification of the source population from extracted cod DNA at 100% (95% CI: 96.4 - 100) and ethanol-preserved fin clip lysates at 100% (95% CI: 88.8 - 100%), excluding the six failed PCR results for the latter sample type. These PCR failures are likely due to ethanol used to preserve these archived fin clip samples. Ethanol is a known PCR inhibitor, and its effect would be prominent when crude lysates were used directly in PCR without DNA purification. The effect of ethanol, however, will be irrelevant to our intended use in real-world testing since these fish samples will not be preserved but tested on-site in real-time. Next, in the experiment demonstrating the workflow of Fish-OLA, we found that cod DNA samples extracted by students and teaching assistants using the Boom method were 100% amplifiable by our newly designed primers (Fig. 1d). Blinded cod samples (Fig. 1e) tested by students and teaching assistants using Fish-OLA kits resulted in a 92% accuracy (95% CI: 84.5 - 96.9%) and 5% failure rate (95% CI: 1.2 - 11.7%, Fig. 1f). In summary, Fish-OLA is a powerful new tool for species and population identification with high portability, low cost, and high performance. OLA-Simple can be adapted to classify other species with known SNP biomarkers. It has a 2-hour sample-to-results timeline and can be operated on a $200 thermal cycler with a US$10 consumable cost per assay.


Acknowledgements (Optional): : We thank our collaborators: Dr. Lisa Frenkel and Ingrid A Beck, from Seattle Children's Research Institute. They have contributed to developing the OLA-Simple HIV drug resistance test, in which we adapted the chemistry to create this assay for these cod species. We thank other Lutz lab members: Samuel Stewart and Leila Ahmadinia, for their technical support. We also thank the students of the SAFS FISH340 course in autumn 2021 for their participation in the experiment. NP was funded by NIH AI145486 (BRL), NSF ECCS-1835847 (KFB), and the University of Washington Center of Environmental Forensic Science (KFB and NP). CT was partially funded by the Cooperative Institute for Climate, Ocean, & Ecosystem Studies (CICOES) under NOAA Cooperative Agreement NA20OAR4320271 and by NOAA Saltonstall Kennedy grant # 16WCR034. The reagents and supplies used in this study were funded by the National Science Foundation ECCS-1835847 (KFB) and the University of Washington Center of Environmental Forensic Science (KFB and NP). The funding organizations did not influence the study design, interpretation, and writing. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its sub-agencies.

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https://www.iuufishing.noaa.gov/RecommendationsandActions/RECOMMENDATION1415/FinalRuleTraceability.aspx.

(2) Spies, I.; Spurr, R.; Hartinger, T.; Drinan, D.; Tarpey, C.; Hauser, L. Diversifying selection and spatial patterns in the zona
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