Effects of Poor Sanitation Procedures on Cross-Contamination of Animal Species in Ground Meat Products

The presence of \u3c1% of an undeclared species in ground meat is generally thought to be indicative of cross-contamination as opposed to intentional mislabeling; however, this has not been experimentally tested. The objective of this study was to quantify the effects of poor sanitation on the cross-contamination of animal species in ground meat products, with the example of undeclared pork in ground beef. Cross-contamination was quantified using real-time polymerase chain reaction (PCR). Three different sanitation treatments were tested with a commercial grinder (“no cleaning”, “partial cleaning”, or “complete cleaning”) in between grinding of pork and beef samples (13.6 kg each). A 100-g sample was collected for each 0.91 kg (2 lb) of beef processed with the grinder and each sanitation treatment was tested twice. For the “no cleaning” treatment, the first 100-g sample of ground beef run through the grinder contained 24.42 ± 10.41% pork, while subsequent samples (n = 14) contained \u3c0.2% pork. With “partial cleaning,” the first sample of ground beef contained 4.60 ± 0.3% pork and subsequent samples contained \u3c0.2% pork. Pork was not detected in ground beef following “complete cleaning.” These results indicate that incomplete cleaning of grinding equipment leads to species cross-contamination at levels of \u3c1% in most cases. Proper sanitation procedures must be followed when grinding multiple species in order to prevent cross-contamination and product mislabeling

Identification of Species in Ground Meat Products Sold on the U.S. Commercial Market Using DNA-Based Methods

The objective of this study was to test a variety of ground meat products sold on the U.S. commercial market for the presence of potential mislabeling. Forty-eight ground meat samples were purchased from online and retail sources, including both supermarkets and specialty meat retailers. DNA was extracted from each sample in duplicate and tested using DNA barcoding of the cytochrome c oxidase I (COI) gene. The resulting sequences were identified at the species level using the Barcode of Life Database (BOLD). Any samples that failed DNA barcoding went through repeat extraction and sequencing, and due to the possibility of a species mixture, they were tested with real-time polymerase chain reaction (PCR) targeting beef, chicken, lamb, turkey, pork and horse. Of the 48 samples analyzed in this study, 38 were labeled correctly and 10 were found to be mislabeled. Nine of the mislabeled samples were found to contain additional meat species based on real-time PCR, and one sample was mislabeled in its entirety. Interestingly, meat samples ordered from online specialty meat distributors had a higher rate of being mislabeled (35%) compared to samples purchased from a local butcher (18%) and samples purchased at local supermarkets (5.8%). Horsemeat, which is illegal to sell on the U.S. commercial market, was detected in two of the samples acquired from online specialty meat distributors. Overall, the mislabeling detected in this study appears to be due to either intentional mixing of lower-cost meat species into higher cost products or unintentional mixing of meat species due to cross-contamination during processing

Authentication of Red Snapper (\u3cem\u3eLutjanus campechanus\u3c/em\u3e) Fillets Using a Combination of Real-time PCR and DNA Barcoding

Red snapper (Lutjanus campechanus) is a historically overfished and highly valued species that is commonly substituted with other fish, such as tilapia, rockfish, and other snapper species. The objective of this study was to assess the ability of real-time PCR to be used as a screening tool to rapidly test commercial fillets for the presence of red snapper, followed by species identification of negative samples with DNA barcoding. A total of 24 frozen, fresh, or thawed (previously frozen) fillets labeled as “red snapper” were tested with real-time PCR, along with 54 fillets from fish that are common substitutes for red snapper. Real-time PCR parameters were optimized to reduce cross-reactivity. All samples were also tested with DNA barcoding to confirm the identity of fish species. Among the 78 total samples, 3 were authenticated as red snapper with DNA barcoding and successfully detected with real-time PCR. An additional two samples were initially identified as red snapper with real-time PCR but confirmed negative with DNA barcoding, resulting in a false positive rate of 2.7%. Overall, 39.7% of all samples and 91.7% of “red snapper” samples were mislabeled. Red snapper was substituted with other snapper species, rockfish, sea bream, and mahi-mahi. These results illustrate the ability of real-time PCR to be used as a screening tool and the importance of species confirmation with DNA barcoding. Real-time PCR has the potential to be used as a rapid on-site screening tool for regulatory and industry officials to determine the authenticity of red snapper fillets

Use of the Mitochondrial Control Region as a Potential DNA Mini-Barcoding Target for the Identification of Canned Tuna Species

In this study, a DNA mini-barcoding methodology was developed for the differentiation of species commonly found in canned tuna. Primers were designed to target a 236-base pair (bp) fragment of the mitochondrial control region (CR) and a 179-bp fragment of the first internal transcribed spacer region (ITS1). Phylogenetic analysis revealed the ability to differentiate 13 tuna species on the basis of the CR mini-barcode, except in a few cases of species introgression. Supplementary use of ITS1 allowed for differentiation of introgressed Atlantic bluefin tuna (Thunnus thynnus) and albacore tuna (Thunnus alalunga), while differentiation of introgressed Atlantic bluefin tuna and Pacific bluefin tuna (Thunnus orientalis) requires a longer stretch of the CR. After primer design, a market sample of 53 commercially canned tuna products was collected for testing. This mini-barcoding system was able to successfully identify species in 23 of the products, including albacore tuna, yellowfin tuna (Thunnus albacares), and skipjack tuna (Katsuwonus pelamis). One instance of mislabeling was detected, in which striped bonito (Sarda orientalis) was identified in a product labeled as tongol tuna (Thunnus tonggol). PCR amplification and sequencing was unsuccessful in a number of products, likely due to factors such as the presence of PCR inhibitors and DNA fragmentation during the canning process. Overall, CR and ITS1 show high potential for use in identification of canned tuna products; however, further optimization of the assay may be necessary in order to improve amplification and sequencing success rates

Identification of Species in Commercially Sold Game Meats using DNA Barcoding

Game meats represent a multibillion dollar industry in the United States with high economic incentives associated with species substitution and mislabeling. However, there is currently a lack of information regarding the prevalence of mislabeled game meat on the U.S commercial market. The purpose of this study was to conduct a market survey of whole-cut game products sold within the United States to identify incidences of mislabeling using DNA barcoding. Identified species were also examined for classification as a threatened or endangered species. Fifty-four whole-cut game meat samples were collected from online distributors in the United States and sequenced across the 658 base-pair region of the cytochrome c oxidase subunit I (COI) gene. The sequenced DNA was identified based on top species matches in the Barcode of Life Database (BOLD) and GenBank. Data analysis revealed 18.5% of samples were mislabeled and 9.3% of samples were from a near-threatened or vulnerable species. Mislabeled game products included bison and yak identified as domestic cattle, red deer identified as llama and alpaca and black bear identified as beaver. Mislabeled products appeared to have been misbranded for economic gain or due to product mishandling. Although near threatened (bison) and vulnerable (lion) species were identified, the products were correctly labeled by the distributor. The results of this study revealed mislabeled game meat on the U.S. commercial market and suggest the need for further investigation of incidences to identify trends and prevalence

Use of DNA Barcoding Combined with PCR-SFLP to Authenticate Species in Bison Meat Products

American bison (Bison bison) meat is susceptible to species mislabeling due to its high value and similar appearance to meat from domestic cattle (Bos taurus). DNA barcoding is commonly used to identify animal species. However, as a result of the historical hybridization of American bison and domestic cattle, additional genetic testing is required for species confirmation. The objective of this study was to perform a market survey of bison meat products and verify the species using DNA barcoding combined with polymerase chain reaction-satellite fragment length polymorphism (PCR-SFLP). Bison products (n = 45) were purchased from a variety of retailers. Samples that were positive for domestic cattle with DNA barcoding were further analyzed with PCR-SFLP. DNA barcoding identified bison in 41 products, red deer (Cervus elaphus) in one product, and domestic cattle in three products. PCR-SFLP confirmed the identification of domestic cattle in two samples, while the third sample was identified as bison with ancestral cattle DNA. Overall, mislabeling was detected in 3 of the 45 samples (6.7%). This study revealed that additional DNA testing of species that have undergone historical hybridization provides improved identification results compared to DNA barcoding alone

Concentration of Listeria monocytogenes in Skim Milk and Soft Cheese through Microplate Immunocapture

Microplate immunocapture is an inexpensive method for the concentration of foodborne pathogens using an antibody-coated microplate. The objective of this study was to determine the efficacy of microplate immunocapture as an alternative to traditional enrichment for concentrating Listeria monocytogenes to levels detectable with selective plating or real-time PCR. L. monocytogenes isolates serologically characterized as Type 1 (1/2a) and Type 4 (untypeable) were grown overnight and diluted to 100 to 106 colony-forming units (CFU)/mL. The isolates were used to optimize microplate immunocapture in tryptic soy broth with 0.6% yeast extract (TSBYE), skim milk, and queso fresco samples. Following microplate immunocapture, the bacteria were streaked onto polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) agar, followed by incubation at 37 °C for 24 ± 2 h. The bacteria also underwent real-time polymerase chain reaction (PCR). The optimized microplate immunocapture method was tested in triplicate for its ability to capture L. monocytogenes in broth and food samples. Overall recovery rates for L. monocytogenes in food samples at cell populations of 100, 102, and 104 CFU/25 g using microplate immunocapture with real-time PCR were 88.9%, 94.4%, and 100%, respectively. Recovery in these matrices using microplate immunocapture with selective plating was comparatively lower, at 0%, 44.4%, and 100%, respectively. Conventional culture method showed 100% detection at each inoculation level. Microplate immunocapture combined with real-time PCR shows high potential to reduce the time required for detection, with concentration of L. monocytogenes to detectable levels within 1–4 h. The incorporation of a short enrichment step may improve recovery rates at low cell levels

Identification of Shark Species in Commercial Products using DNA Barcoding

Sharks are harvested globally and sold in a variety of commercial products. However, they are particularly vulnerable to overfishing and many species are considered protected or endangered. The objective of this study was to identify species in various commercial shark products and to assess the effectiveness of three different DNA barcoding primer sets. Thirty-five products were collected for this study, including fillets, jerky, soup, and cartilage pills. DNA barcoding of these products was undertaken using two full-length primer sets and one mini-barcode primer set within the cytochrome c oxidase subunit (COI) gene. Successfully sequenced samples were then analyzed and identified to the species level using sequence databases and character-based analysis. When the results of all three primer sets were combined, 74.3% of the products were identified to the species level. Mini-barcoding showed the highest success rate for species identification (54.3%) and allowed for a wide range of identification capability. Six of the 26 identified products were found to be mislabeled or potentially mislabeled, including samples of shark cartilage pills, shark jerky, and shark fin soup. Six products contained species listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendices and 23 products contained near-threatened, vulnerable or endangered species according to the International Union for the Conservation of Nature (IUCN) Red List. Overall, this study revealed that a combination of DNA barcoding primers can be utilized to identify species in a variety of processed shark products and thereby assist with conservation and monitoring efforts

Identification of Meat and Poultry Species in Food Products Using DNA Barcoding

DNA barcoding is a promising method for the sequencing-based identification of meat and poultry species in food products. However, DNA degradation during processing may limit recovery of the full-length DNA barcode from these foods. The objective of this study was to investigate the ability of DNA barcoding to identify species in meat and poultry products and to compare the results of full-length barcoding (658 bp) and mini-barcoding (127 bp). Sixty meat and poultry products were collected for this study, including deli meats, ground meats, dried meats, and canned meats. Each sample underwent full and mini-barcoding of the cytochrome c oxidase subunit I (COI) gene. The resulting sequences were queried against the Barcode of Life Database (BOLD) and GenBank for species identification. Overall, full-barcoding showed a higher sequencing success rate (68.3%) as compared to mini-barcoding (38.3%). Mini-barcoding out-performed full barcoding for the identification of canned products (23.8% vs. 19.0% success), as well as for turkey and duck products; however, the primer set performed poorly when tested against chicken, beef, and bison/buffalo. Overall, full barcoding was found to be a robust method for the detection of species in meat and poultry products, with the exception of canned products. Mini-barcoding shows high potential to be used for species identification in processed products; however, an improved primer set is needed for this application

Development of a DNA Mini-Barcoding Protocol Targeting COI for the Identification of Elasmobranch Species in Shark Cartilage Pills

Many elasmobranch (shark and ray) species are considered threatened and their identification in processed products is important for conservation and authentication purposes. However, identification of elasmobranch species in shark cartilage pills has proven difficult using existing methodologies. The objective of this study was to develop a DNA mini-barcoding protocol using a ~130 bp region of the cytochrome c oxidase subunit I (COI) gene for species identification in shark cartilage pills. A total of 22 shark cartilage products underwent DNA extraction in duplicate using the DNeasy Blood and Tissue Kit (Qiagen). The effectiveness of a clean-up step following DNA extraction was analyzed by comparing DNA purity values and polymerase chain reaction (PCR) amplification rates. Next, five different mini-barcode primer sets were compared based on amplification rates, and the three top-performing primer sets were used in DNA sequencing. The incorporation of a clean-up step following DNA extraction showed a slight advantage over DNA extraction alone, with a higher amplification rate (52.3% vs. 47.8%) and A260/A230 value (3.3 vs. 0.6). The three primer sets selected for DNA mini-barcoding showed DNA sequencing rates of 54.5–65.9% among the 44 duplicate samples. When the results for all three primer sets were combined, 18 of the 22 shark cartilage products were identified to the species or genus level. On an individual basis, the best-performing primer set identified 16 of the 22 products to the species or genus level. Overall, the protocol developed in this study increased the identification rate for elasmobranches in cartilage products by more than 2-fold as compared to previous research