Purpose: This work is part of a project to validate identification of crustacean shellfish using CO1 barcoding.
Methods: A ≈655 bp fragment of the CO1 gene was amplified using PCR, and products were sequenced in both forward and reverse directions using the Sanger method. Sequences were aligned and used to generate neighbor-joining consensus trees. In addition to assessing intra- and inter-species variation versus sequencing (run to run) variation, we optimized PCR and determined the effects of sample handling and storage.
Results: Adverse sample handling, such as multiple freeze-thaw cycles, were found to adversely affect CO1 PCR results for some products. This is in contrast to previous experience with vertebrate fish tissues. In nearly all tests of inter- and intra-species variation, we obtained bidirectional sequence reads at least 650 bp in length with 98-100% high quality base calls. Results indicate that the method is able to effectively differentiate most commercial crustacean species and successfully identify unknowns. A library of reference standard COI sequences is also being developed.
Significance: DNA barcoding has proven to be an accurate and reliable method for species identification of processed and packaged seafood which cannot be identified through standard morphological means. Species identification is important for both protecting food safety and detecting seafood fraud. Our work indicates that CO1 DNA barcoding, previously proven to be a useful tool for the identification of vertebrate fish species, will also be a valuable tool for identifying commercial crustacean shellfish.