A Multiplex PCR Method for the Identification of Commercially Important Salmon and Trout Species (Oncorhynchus and Salmo) in North America
ABSTRACT The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5′ cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) “barcode” sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats.
Practical Application: This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.
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- "DNA barcoding can be usedt oi dentify specimens including whole fish, fillets, fins, juveniles, larvae, eggs, or tissue fragments. It is recognized by the Food and Drug Administration in the USAa sa replacement for the time-consuming technique of proteini soelectric focusing forfi sh identification (Yancye ta l. 2008; Handy et al. 2011) and can be applied to raw,c ooked( Wong and Hanner 2008),o r smokedfi sh (Smith et al. 2008; Millera nd Mariani 2010).Italso has the potential to be used with heavily processed food samplesb yu sing shortm ini barcode regions (reviewed in Rasmussen et al. (2009)), andthe referencel ibrary aids constructiono fm olecular probes basedo ns hort, species-specific patterns of variation found in the standard barcode sequence (Eytan and Hellberg 2010; Rasmussen et al. 2010). The potential of DNA barcoding to provide unequivocals pecies assignments from wholeo rp artial specimens may significantly reforms eafoodm arket practices, particularly for commercially important species (Rasmussen et al. 2009). "
ABSTRACT: The Fish Barcode of Life campaign involves a broad international collaboration among scientists working to advance the identification of fishes using DNA barcodes. With over 25% of the world's known ichthyofauna currently profiled, forensic identification of seafood products is now feasible and is becoming routine. Driven by growing consumer interest in the food supply, investigative reporters from five different media establishments procured seafood samples (n = 254) from numerous retail establishments located among five Canadian metropolitan areas between 2008 and 2010. The specimens were sent to the Canadian Centre for DNA Barcoding for analysis. By integrating the results from these individual case studies in a summary analysis, we provide a broad perspective on seafood substitution across Canada. Barcodes were recovered from 93% of the samples (n = 236), and identified using the Barcode of Life Data Systems "species identification" engine ( www.barcodinglife.org ). A 99% sequence similarity threshold was employed as a conservative matching criterion for specimen identification to the species level. Comparing these results against the Canadian Food Inspection Agency's "Fish List" a guideline to interpreting "false, misleading or deceptive" names (as per s 27 of the Fish Inspection regulations) demonstrated that 41% of the samples were mislabeled. Most samples were readily identified; however, this was not true in all cases because some samples had no close match. Others were ambiguous due to limited barcode resolution (or imperfect taxonomy) observed within a few closely related species complexes. The latter cases did not significantly impact the results because even the partial resolution achieved was sufficient to demonstrate mislabeling. This work highlights the functional utility of barcoding for the identification of diverse market samples. It also demonstrates how barcoding serves as a bridge linking scientific nomenclature with approved market names, potentially empowering regulatory bodies to enforce labeling standards. By synchronizing taxonomic effort with sequencing effort and database curation, barcoding provides a molecular identification resource of service to applied forensics.Mitochondrial DNA 10/2011; 22 Suppl 1(S1):106-22. DOI:10.3109/19401736.2011.588217 · 1.70 Impact Factor
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ABSTRACT: This interlaboratory study evaluated a real-time multiplex polymerase chain reaction (PCR) method for identification of salmon and trout species in a range of commercial products in North America. Eighty salmon and trout products were tested with this method by three independent laboratories. Samples were collected in the United States and Canada, and only the collecting institution was aware of the species declaration. Following analysis with real-time PCR, all three laboratories were able to identify species in 79 of the 80 products, with 100% agreement on species assignment. A low level of fraud was detected, with only four products (5%) found to be substituted or mixtures of two species. The results for two of the fraudulent products were confirmed with alternate methods, but the other two products were heavily processed and could not be verified with methods other than real-time PCR. Overall, the results of this study show the usefulness and versatility of this real-time PCR method for the identification of commercial salmon and trout species.Journal of Agricultural and Food Chemistry 02/2011; 59(3):876-84. DOI:10.1021/jf103241y · 3.11 Impact Factor
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ABSTRACT: Wildlife crime enforcement is increasingly relying on genetic techniques to deliver forensic evidence to investigators. Forensic DNA applications require robust molecular markers, informative at the level of the species, population and individual, in a wide range of taxa. Within species, single-nucleotide polymorphisms (SNPs) have long been recognized as offering many potential advantages over traditional microsatellite markers; however, the best methods for their discovery, validation and genotyping in non-model species remain an area of novel research and much discussion. The potential availability to wildlife geneticists of deep sequencing platforms and high-density genotyping arrays appears to promise an almost infinite source of variable markers for determining the geographic or individual origin of a sample. This study examines the drivers for developing SNP genotyping panels in wildlife forensics and their potential as applied tools, before examining a range of strategies that are being employed to try to unlock this potential and address current questions in wildlife conservation and enforcement.Molecular Ecology Resources 03/2011; 11 Suppl 1:109-16. DOI:10.1111/j.1755-0998.2010.02954.x · 5.63 Impact Factor