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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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 1:106-22. · 1.71 Impact Factor
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ABSTRACT: There is increasing demand to develop cost effective and accurate approaches to analysing biological tissue samples. This is especially relevant in the fishing industry where closely related fish samples can be mislabelled, and the high market value of certain fish leads to the use of alternative species as substitutes. For example Barramundi and Nile Perch (belonging to the same genus, Lates). There is a need to combine selective proteomic data sets with sophisticated computational analysis to devise a robust classification approach. This paper describes an integrated mass spectrometry based proteomics and bioinformatics approach to classifying a range of fish samples. A classifier is developed using training data that successfully discriminates between Barramundi and Nile Perch samples using a selected protein subset of the proteome. Additionally, the classifier is shown to successfully discriminate between test samples not used to develop the classifier, including samples that have been cooked, and to classify other fish species as neither Barramundi nor Nile Perch. This approach has applications to truth in labelling for fishmongers and restaurants, monitoring fish catches, and for scientific research into distances between species. This article is protected by copyright. All rights reserved.Proteomics 09/2013; · 4.43 Impact Factor
Article: Fish frauds: the DNA challenge[Show abstract] [Hide abstract]
ABSTRACT: The rising demand for seafood and trade globalization has brought about a rapid increase in the number of fish species traded. Consequently, the occurrence of mislabelling is growing as well, reaching levels of concerns in USA, Canada and Europe. In this light, the evolving consciousness of consumers and the new exigencies of commerce call for greater safety and quality requirements. These factors have made urgent the need for efficient traceability systems, aimed to ensure transparency on the identity and origin of the traded products and the compliance with the regulations concerning illegal fisheries and labelling. Moreover, greater efforts are necessary to create a list of market names that can be recognized both locally and internationally, in order to overcome the confusion regarding fish denominations. In this context, molecular analysis represents the most promising challenge to verify and support traceability in the seafood chain. Nowadays, the three mitochondrial genes cytb, COI and 16srRNA are the most targeted for this purpose and, among the available procedures, the DNA bar coding is the most commonly applied to verify the labelling compliances, also at the official level. In this review, the most important issues relating to these topics have been reported and discussed.CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources 10/2012; 7(71).