Performing Department
(N/A)
Non Technical Summary
Ranked as one of the world's most valuable commercial seafood, tuna species belonging to the Thunnus genus are considered the most important and endangered fish in worldwide trade. Extensive research to develop methods to distinguish Thunnus at the species level has significantly contributed to the enormous databanks of genetic information on these fish. However to date, no single approach has been successful in completely discriminating all the Thunnus species. Applied Food Technologies (AFT) has been successful in the development of molecular diagnostics for fish species identification. Our two most common approaches include PCR multiplexing utilizing species specific primers and DNA barcoding based on the COI gene sequence alignments. However as others, we have shown that neither approach is specific enough to distinguish these closely related species. A number of other methods based on RFLPs, RAPDs, SSCPs or AFLPs as well as sequencing with numerous different gene targets have also not been successful in distinguishing all species in the Thunnus genus. In Phase I of this project we targeted a different gene sequence to the COI gene that can be utilized to distinguish the six most popular Thunnus species in the fresh form and designed and tested primers that are unique to three of the six species. Success achieved in Phase I feasibility study has positioned AFT, in Phase II, to focus research and development efforts on final optimization of molecular diagnostic methods developed in Phase I and integrate additional commercially important species that are sold under the "tuna" label. Once optimized for fresh/frozen tuna, we will investigate modifications in DNA extraction protocols to enable the use of the diagnostic to identify tuna species in further processed tuna products (canned and in pouches). Our overarching goal is to deliver a commercialized multiplex diagnostic test that will readily distinguish the eight major, high-valued tuna species in commerce from one another as well as from the commonly substituted species found in commerce. The global potential of this research will serve more broadly as a template for the regulatory management of other species with close evolutionary relatedness that are also found subject to fraudulent labeling practice by providing regulators and the industry with a powerful set of tools enabling species identification and authentication. To date, regulators monitoring product labels in commerce and fisheries management groups do not have an available tool to distinguish all Thunnus species. The diagnostic developed as part of this USDA SBIR will be commercially available in our current species identification testing service as well as for use in Federal, State, public and private laboratories. This work also has potential to serve as platform technology in developing DNA-based methods for distinguishing other genetically close related species.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
The long-term goal of this project is to develop a reproducible, cost-effective molecular diagnostic that will unambiguously identify and distinguish the major commercially traded tuna species, fresh frozen and canned. While ongoing programs at AFT as well as efforts in several academic and regulatory groups continue to extend our capabilities to speciate many commercially important fish, the genetic complexity and relatedness of the major tuna species has presented significant challenges in developing a definitive diagnostic for fish of the Thunnus genus. The technical objectives of this project are to procure and authenticate reference specimens representing the additional tuna and tuna substitute species; complete design and testing of species specific primers for differentiating all genetic-related tuna species and their common substitute; test primer/probe tuna species specific strategy on real time PCR platforms; and optimize an extraction protocol for canned tuna and test with newly designed primers and probes. The final objective is to validate the lead DNA-based species identification method for both fresh/frozen and canned tuna products. The current FDA recommended protocol for DNA-based fish species identification is COI DNA barcoding, which does not differentiate all tuna species from one another. Successful development and implementation of the deliverable to be produced in this proposed project will provide a species authentication tool of significant value to seafood distributors, processors, and Federal and state regulatory agencies that is currently not available for tuna. This model system has the potential to serve as the platform technology for developing testing for other groups of closely related fish species not readily discriminated by current methods but also for other markets in the food industry dealing with issues of fraudulent labeling, traceability, species substitution as well as stock assessment and species conservation.
Project Methods
Most critical in designing accurate and defensible species identification diagnostics for tuna is to authenticate and professionally validate reference material. Working with our industry collaborators and Federal and State agencies, additional fish species to complete our current validated tuna species collection will be procured and taxonomically identified. A series of species specific primers targeting mitochondrial genes for discriminating between the six Thunnus species were designed and tested in Phase I. For the additional tuna and related species added in Phase II, comparable species specific primers will be designed and alternate gene targets will be used if necessary for ensuring discrimination among all the targeted fish species. Primer specificity for intended target will be confirmed on real time PCR platform and performance of all primers will be optimized. The universal primer set for a species-specific probe strategy designed and tested in Phase I, will be tested on the additional tuna and related species template to be included in this project. Primer/probe combinations for all tunas will initially be tested in singleplex real time PCR before converting and optimizing reactions to a multiplex format. We anticipate 3 multiplex reactions will definitively differentiate all eight highly related tunas as well as a commonly substituted species in trade. Species identification of canned and highly processed tuna remains challenging due to compromised quality of its DNA. The DNA from canned tuna will be isolated utilizing commercially available extraction kits to identify the optimal protocol that yields DNA template of quality and quantity sufficient for reproducible downstream amplification. Efforts will be made to design the most streamlined protocol that is compatible with efficient, cost-effective screening for canned tuna samples. Reproducibility of our DNA diagnostic platforms must be rigorously challenged to establish the robust nature of these methods for use in our in-house tuna species authentication test and in outside laboratory tests. We will standardize all components and methodology of the lead tuna species I.D. diagnostic to ensure reproducible results. Following proper sourcing of authenticated tuna species, we will conduct double-blind studies for ensuring accuracy of the diagnostic. We recognize the inherent variability of PCR platforms and thus will run the test for consistency using several real time PCR thermocyclers, from various manufactures with the procedure being executed by a number of technical personnel from different laboratories. Finally, independent and certified labs will conduct third party validation of the diagnostic procedures.