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.

本研究量化了北卡罗来纳州沿海与内陆地区虾类产品的标签误标程度。本研究遵循凯杰公司（Quiagen, INC）的DNAEasy提取流程，从约20mg的虾类组织中提取基因组DNA。为实现样本的物种水平鉴定，本研究聚焦于线粒体DNA细胞色素氧化酶I基因（mitochondrial DNA cytochrome oxidase I gene, CO1）的测序；该基因已被应用于多项海鲜标签误标相关研究（如Cox等，2013；Staffen等，2017；Willette等，2017）。本研究参考Willette等（2017）的聚合酶链式反应（Polymerase Chain Reaction, PCR）流程，以及Ivanova等（2009）的引物混合液体系，对提取的DNA进行CO1序列扩增。为制备25μL的PCR反应体系，我们将DNA、CO1_F1、CO1_F2、CO1_R1及CO1_R2引物混合液、去离子水，与包含所有必需PCR反应组分的PuRe Taq Ready-To-Go PCR磁珠进行混合。在热循环仪中，样本历经35个循环：95℃变性、50℃退火及70℃延伸。设置不含DNA的全组分PCR阴性对照，以检测实验污染情况。通过1%琼脂糖凝胶电泳检测PCR扩增产物，以判断DNA扩增是否成功。扩增出约650碱基对条带的样本，被送往北卡罗来纳州罗利的ETON Bioscience实验室进行测序。将成功测序区域的峰图，与美国国家生物技术信息中心（National Center for Biotechnology Information, NCBI）核苷酸序列数据库GenBank中已知样本的CO1序列，通过基本局部比对搜索工具（Basic Local Alignment Search Tool, BLAST）进行比对。仅当序列一致性百分比与查询覆盖度均大于等于98%，且e值趋近于零时，方可确定样本的物种分类。经鉴定为*Litopenaeus setiferus*的样本视为北卡罗来纳州本地虾类；而鉴定为*Litopenaeus vannamei*的样本则为太平洋白对虾，属于标签误标产品。