PCR Cloning Combined With DNA Barcoding Enables Partial Identification of Fish Species in a Mixed-Species Product

Abstract

DNA barcoding is a valuable tool for regulatory identification of fish species; however, it does not perform well when multiple species are present within the same food product. Therefore, the objective of this study was to examine the use of PCR cloning 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). Three subsamples from each fish ball underwent DNA extraction, full DNA barcoding (655 bp), and mini-barcoding (226 bp) of the cytochrome c oxidase subunit 1 (CO1) gene. Subsamples that did not pass sequencing according to regulatory standards were further analyzed with PCR cloning. All fish balls made of just one species tested positive for that species (i.e., tilapia, cod, or pollock) with both full and mini-barcoding. However, only tilapia was detected in fish balls containing multiple species when tested with standard barcoding techniques, reflecting an inaccurate representation of the fish mixture and suggesting species bias. PCR cloning allowed for identification of Pacific cod in 86% of the mixed-species fish balls tested with fullbarcode cloning and 100% of the mixed-species fish ball tested with mini-barcode cloning. However, PCR cloning did not enable the identification of walleye pollock. Standard full barcoding produced more high quality sequences compared to minibarcoding yet failed to accurately detect all species present in the tested fish mixtures. 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 overcome the species bias observed in this study.

A case study on DNA barcoding for pet food mislabeling in South Korea

“…In the present study, DNA barcoding was applied to investigate mislabeling in different pet food products, including canned meats, jerky, particle, and snacks. The available reports demonstrated that DNA barcoding is a promising approach for detecting fish, meat and poultry species in food products (Handy et al, 2011;Armani et al, 2015;Silva et al, 2020). Moreover, combining PCRs and cloning targeting a barcode region may be an effective method to determine species from mixed-species products (Silva et al, 2020).…”

“…The available reports demonstrated that DNA barcoding is a promising approach for detecting fish, meat and poultry species in food products (Handy et al, 2011;Armani et al, 2015;Silva et al, 2020). Moreover, combining PCRs and cloning targeting a barcode region may be an effective method to determine species from mixed-species products (Silva et al, 2020). However, DNA degradation during food processing may restrict the recovery of barcode sequences from examined effects.…”

“…Therefore, the application of macro and minibarcoding should be considered, depending on tested products and the quality of extracted DNA. Compared to minibarcoding, full length DNA barcoding was more effective for species identification in meat and poultry products, except for canned samples (Silva et al, 2020). With the increasing number of sequences deposited in the public databases for references such as GenBank and BOLD, DNA barcoding is still a promising method for pet food authentication, especially for single-species products with high-quality extracted DNA.…”

“…Finally, even though PCR, cloning and different primer sets were applied, undetected species in mixed samples due to PCR bias could not be excluded. Bias in the PCR procedure for a specific species can be derived from factors such as primer bias and differences in mitochondrial copy number (Silva et al, 2020). Therefore, further studies should apply macrobarcoding to authenticate mixed pet food products in South Korea.…”