Marketplace Shrimp Mislabeling in North Carolina

We quantified the extent of shrimp mislabeling in coastal and inland North Carolina. Following the DNAEasy extraction protocol (Quiagen, INC), we extracted genomic DNA from approximately 20mg shrimp tissue. To identify individual samples to the species-level we focused on sequencing the mitochondrial DNA cytochrome oxidase I gene (CO1). It has been used in other seafood mislabeling studies (e.g., Cox et. al 2013, Staffen et al. 2017, Willette et. al 2017). We amplified CO1 sequences from extracted DNA following the PCR protocol outlined in Willette et al. (2017) and a primer cocktail from Ivanova et al. (2009). To prepare a 25μL sample for PCR, we combined the DNA with a primer cocktail of CO1_F1, CO1_F2, CO1_R1, and CO1_R2, deionized water, and a PuRe Taq Ready-To-Go PCR bead containing the necessary PCR components. In the thermal cycler, the samples went under 35 cycles of 95°C for denaturation, 50°C for annealing, and 70°C for extension. A negative control containing all of the PCR components, except DNA, was used to test for contamination. We ran the PCR products on a 1% agarose gel to determine if PCR amplification of the DNA was successful. Samples with successful ~650 base pair bands were sent to an ETON Bioscience facility in Raleigh, NC for sequencing. Chromatograms of successfully sequenced regions were then matched against CO1 sequences of known samples on National Center for Biotechnology Information’s nucleotide collection database GenBank using the Basic Local Alignment Search Tool (BLAST). We only concluded the identity of a species if the percent identity and query coverage was greater than or equal to 98% and the e-value was close to zero. Samples identified as <i>Litopenaeus setiferus</i> were considered to be local North Carolina shrimp while <i>Litopenaeus vannamei</i> samples were determined to be Pacific whiteleg shrimp, and thus mislabeled.