Date of Award
Master of Science (MS)
Dr. Rosalee Hellberg
Dr. Michael Kawalek
Dr. Anuradha Prakash
DNA barcoding is a valuable tool for fish species identification by food regulators, however, it does not perform well when multiple species are present within the same food product. PCR cloning has high potential to be used in combination with DNA barcoding to overcome this challenge. The objective of this study was to examine the use of PCR cloning combined with DNA barcoding to identify fish in a mixed-species product that cannot be identified with standard DNA barcoding. A total of 15 fish ball mixtures were prepared with known amounts of Nile tilapia (Oreochromis niloticus), Pacific cod (Gadus macrocephalus), and walleye pollock (Gadus chalcogrammus). The fish balls underwent DNA extraction in triplicate, followed by DNA barcoding across the full barcode (655 bp) and SH-E mini-barcode (226 bp) of the cytochrome c oxidase subunit 1 (CO1) region. Samples that did not pass sequencing according to regulatory standards were further analyzed with PCR cloning. Full barcoding enabled identification of at least one species in 80% of the fish ball mixtures compared to 51% for minibarcoding. The results of PCR cloning with samples that did not pass DNA barcoding showed identification success rates of 61% for clones (54 of 90) that underwent full barcoding and 51% for clones (111 of 220) that underwent mini-barcoding. All fish balls made of just one species tested positive for that species (i.e., tilapia, cod, or pollock).. The combination of standard full barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 6 of 12 (50%) of mixed-species fish balls. In comparison, the combination of standard mini-barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 9 of 12 (75%) of mixed-species fish balls. Overall, the results of this study show that PCR cloning may be an effective method to identify certain fish in mixed-species products when standard DNA barcoding fails. However, additional research is needed to understand the limitations associated with primer bias.
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Silva, A. (2019). Use of PCR cloning combined with DNA barcoding to identify fish in a mixed-species product. Master's thesis, Chapman University. https://doi.org/10.36837/chapman.000065