Validation of FASTFISH-ID: A new commercial platform for rapid fish species authentication via universal closed-tube barcoding.

Seafood represents up to 20% of animal protein consumption in global food consumption and is a critical dietary and income resource for the world's population. Currently, over 30% of marine fish stocks are harvested at unsustainable levels, and the industry faces challenges related to Illegal, Unregulated and Unreported (IUU) fishing. Accurate species identification is one critical component of successful stock management and helps combat fraud. Existing DNA-based technologies permit identification of seafood even when morphological features are removed, but are either too time-consuming, too expensive, or too specific for widespread use throughout the seafood supply chain. FASTFISH-ID is an innovative commercial platform for fish species authentication, employing closed-tube barcoding in a portable device. This method begins with asymmetric PCR amplification of the full length DNA barcode sequence and subsequently interrogates the resulting single-stranded DNA with a universal set of Positive/Negative probes labeled in two fluorescent colors. Each closed-tube reaction generates two species-specific fluorescent signatures that are then compared to a cloud-based library of previously validated fluorescent signatures. This novel approach results in rapid, automated species authentication without the need for complex, time consuming, identification by DNA sequencing, or repeated analysis with a panel of species-specific tests. Performance of the FASTFISH-ID platform was assessed in a blinded study carried out in three laboratories located in the UK and North America. The method exhibited a 98% success rate among the participating laboratories when compared to species identification via conventional DNA barcoding by sequencing. Thus, FASTFISH-ID is a promising new platform for combating seafood fraud across the global seafood supply chain.

Dimethyldimethyl Hydantoin: An Alternative Fluid for Morphological and Genetic Preservation.

Modern taxonomy requires the preservation of biospecimens for both morphological and molecular applications. The utility of a previously identified preservative, dimethyldimethylhydantoin hydantoin (Dekafald®), to retain both physical diagnostic traits and the DNA integrity of biological specimens remains unknown. Using 439 eggs and 414 larvae from two North American fish species, we compared three hydantoin solutions at different concentrations (5%, 10%, and 20%) with gold standard preservatives (10% buffered formalin, 95% ethanol) to evaluate morphological trait retention up to 90 days, and DNA barcoding success up to 56 days. While the 5% hydantoin solution had the most sequencing success by 56 days, the 10% hydantoin solution was the best multipurpose preservative. Future work should assess the performance of ∼10% hydantoin solution over longer time periods, and its applicability to other taxa such as Arthropoda.

Re-visiting the occurrence of undeclared species in sausage products sold in Canada.

Meat and poultry are major protein sources for humans worldwide. Undeclared ingredients in processed meat products, like sausage, continue to be identified in retail products all over the world. In collaboration with the Canadian Food Inspection Agency, a previous study of products purchased in Canada showed 20% mislabelling rate in sausage meats when tested for beef, pork, chicken, turkey and horse using DNA barcoding and digital PCR. In a follow-up to this study, an additional 100 "single species" sausage products were collected from Canadian retail markets, one year after our earlier study, to determine the prevalence of undeclared meat species in sausage. A new hierarchy of complementary molecular methods was applied in this study, including the testing of new target species (sheep and goat), in addition to beef, pork, chicken, turkey and horse. First, all samples were tested using DNA barcoding using universal primers, which revealed that 97% of the samples contained the declared species, presumably as the predominant species. Second, all samples were tested using ddPCR assays specifically targeting beef, pork, chicken, and turkey, which revealed that five beef samples, three chicken samples and two turkey samples contained undeclared species. Additionally, ddPCR revealed the presence of undeclared sheep in five samples. Overall, using complementary molecular methods, 14% of the samples contained additional undeclared species. It was encouraging to find a reduced rate of mislabelling compared to the previous study, though it remains clear that meat mislabelling is still an issue affecting Canadian consumers. The results from this study can be used to support decision-making processes for future inspection and monitoring activities in order to control species substitution or adulteration to protect consumers.

Interlaboratory evaluation of a real-time multiplex polymerase chain reaction method for identification of salmon and trout species in commercial products.

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.