Molecular identification of five commercial flatfish species by PCR–RFLP analysis of a 12S rRNA gene fragment

Refrigerated or frozen fillets of commercial flatfish species are sometimes mislabelled, and identification of those products is needed to avoid fraudulent substitution. Molecular identification of five commercial flatfish species (order Pleuronectiformes), ie Lepidorhombus whiffiagonis (megrim), Platichthys flesus (flounder), Reinhardtius hippoglossoides (Greenland halibut), Scophthalmus maximus (turbot) and Solea vulgaris (= S solea) (sole), has been carried out on the basis of the amplification of an approximately 433 bp segment from the mitochondrial 12S rRNA gene using the polymerase chain reaction (PCR) and universal primers. Direct DNA sequencing from two PCR products for each flatfish species was carried out, and sequences were used to select six restriction enzymes. PCR products of 15 individuals of each species were cut with each enzyme, resulting in species‐specific restriction fragment length polymorphism (RFLP). The five flatfish species could be identified by application of the restriction enzyme AluI as well as by using different combinations of a pair of enzymes, ie DdeI and either AciI or MwoI. No intraspecific genetic polymorphism was found for any of the six enzymes. Results confirmed the usefulness of this technique to distinguish and genetically characterise refrigerated or frozen pieces of these five flatfish species. Copyright © 2003 Society of Chemical Industry     

Genetic differentiation between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR–RFLP analysis of a 12S rRNA gene fragment

PCR–RFLP analysis was applied to the identification of two closely related flatfish species: sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Amplification of DNA isolated from fish muscle samples was carried out using a set of primers flanking a region of 321 base pairs (bp) from the mitochondrial 12S rRNA gene. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for AciI and MwoI endonucleases. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with AciI and MwoI enzymes allowed the unequivocal identification of sole and Greenland halibut species. © 2000 Society of Chemical Industry     

Indirect enzyme-linked immunosorbent assay for the identification of sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides).

Polyclonal antibodies produced against soluble muscle protein extracts from sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides) were used in an indirect enzyme-linked immunosorbent assay for the specific identification of fillets from these flatfish species. The assay was performed in two different formats: microtiter plates and immunostick tubes. Immunorecognition of antibodies adsorbed to their specific fish samples was made with goat antirabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymatic conversion of the substrate allowed unequivocal identification of all flatfish species studied.     

Identification of Flatfish Species Using Polymerase Chain Reaction (PCR) Amplification and Restriction Analysis of the Cytochrome b Gene

Restriction site analysis of PCR products from a conserved region of the cytochrome b gene has been used for the specific identification of sole ( Solea solea ), European plaice ( Pleuronectes platessa ) and flounder ( Platichthys flesus). Polymerase chain reaction (PCR) amplification of the cytochrome b gene using universal primers produced a 359 bp fragment in all species analyzed. Digestion of the PCR products with Nci I, Sau 3AI and Hinf I endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of the fish species. This methodology should prove useful for enforcing labeling regulations in the authentication of flatfish species.     

Real-time PCR method applied to seafood products for authentication of European sole (Solea solea) and differentiation of common substitute species

Judged by quality and taste, the European sole (Solea solea) is considered one of the finest flatfish and is, thus, of considerable commercial value. In the present work, a specific fast real-time PCR was developed for the authentication of S. solea, i.e. to distinguish it from other related species and avoid substitution of this species, either deliberately or unintentionally. The method is based on a species-specific set of primers and MGB Taqman probe which amplifies a 116-bp fragment of the internal transcribed spacer 1 (ITS 1) ribosomal DNA region. This assay combines the high specificity and sensitivity of real-time PCR with the rapidity of the fast mode, allowing the detection of S. solea in a short period of time. The present methodology was validated for application to all types of manufactured products for the presence of S. solea, with successful results. Subsequently, the method was applied to 40 commercial samples to determine whether correct labeling had been employed in the market. It was demonstrated that the assay is a useful tool in monitoring and verifying food labeling regulations.     

Puffer fish-based commercial fraud identification in a segment of cytochrome b region by PCR–RFLP analysis

To identify the mislabeled or fraudulently substituted toxic puffer fish in thermally processed fish products, a polymerase chain reaction (PCR) method using restriction sites and sequence analysis has been developed in this study. A 376-bp fragment of the cytochrome b gene was produced after PCR amplification. Fish tissue samples were prepared under autoclaving conditions at 121 °C for 10–90 min at 10 min intervals. DNA fragments could not be detected after 90 min of autoclaving at 121 °C. For PCR product digestion, BsaJ I, Aci I, Hinf I, Taq I, and Sap I endonucleases were used to yield species-specific profiles for the identification of puffer fish species from 60 commercial market samples. Results from this study showed that the restriction fragment length polymorphism technique can be used to identify 17 puffer fish species from commercial products even after severe thermal processing.     

Detection of pig derivatives in food products for halal authentication by polymerase chain reaction –restriction fragment length polymorphism

A method for detection of the presence of pig derivatives in three types of food products—sausages and casings, bread and biscuits—using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene was developed. Genomic DNA of sausages and casings, bread and biscuits were extracted. The genomic DNA from the food products were found to be of good quality for the sausages and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs (bp). However, no genomic DNA was detected from the casing samples and poor quality of genomic DNA was extracted from bread and biscuits. No amplification of mt cyt b gene was produced from bread and biscuit samples. To differentiate between samples, the amplified PCR products were digested with restriction enzyme (RE) BsaJI, resulting in species‐specific RFLP. The cyt b PCR‐RFLP species identification assay gave excellent results for detection of pork adulteration in food products and is a potentially reliable technique to avoid species adulteration or fraudulent species substitution for halal authentication. Copyright © 2006 Society of Chemical Industry     

Use of immunological techniques for detecting species substitution in raw and smoked fish

 Immunological techniques based on double immunodiffusion and immunodotting have been designed to detect the substitution of halibut (Hippoglossus hippoglossus) for sole (Solea solea) fillets. An immunodotting technique has been also developed to differentiate between smoked cod (Gadus morhua) and smoked eel (Anguilla anguilla) fillets. Antisera raised against water-soluble extracts of raw halibut and smoked cod were employed. Antiserum to cod proteins did not show cross-reaction with eel proteins, whereas antiserum to halibut proteins cross-reacted with sole proteins. Species-specific antiserum to halibut proteins was achieved by adsorption on a polymer of sole proteins. These methods are very easy to perform and provide the basis for the development of rapid tests for detecting species substitution in fish products. Received: 18 April 1996/Revised version: 5 July 1996     

Technical note: DNA typing for ovine MHC DRB1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

The ovine major histocompatibilty complex (Ovar) class II DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of 52 Suffolk sheep. Polymerase chain reaction products were characterized by the restriction fragment length polymorphism (RFLP) technique using nine restriction enzymes, RsaI, HaeIII, SacI, SacII, DdeI, NciI, Hin1I, EcoRI, and BstNI, yielding 13 types. Sequencing of cloned PCR products identified 16 Ovar-DRB1 alleles. Collectively, all PCR-RFLP patterns exactly matched those predicted from DNA sequences. These findings strongly indicate that the PCR-RFLP method using a combination of nine restriction endonucleases is a very powerful tool in Ovar typing.     

Detection and identification of marine fish mislabeling in Guangzhou's supermarkets and sushi restaurants using DNA barcoding

In this study, DNA barcoding was applied to identify the distinct species of fish products in Guangzhou supermarkets and sushi restaurants in order to confirm whether products were correctly labeled. Samples were analyzed using mitochondrial cytochrome C oxidase subunit I (CO I) gene as the target. Our results showed that the CO I gene of all 139 samples examined was successfully amplified by PCR. When sequenced, 30 samples (21.58%) were mislabeled as the wrong species, 11 samples had insufficient information provided on the label to determine if the labeling was correct (7.91%), and four samples failed sequencing (2.88%). We also found that the use of proper labels for fish products in sushi restaurants was higher than that in supermarkets. As a simple, rapid, and efficient technology, DNA barcoding can be widely used for species identification of fish products. Our work shows that regulation of the labeling of fish products, as we evaluated in Guangzhou and other markets in China, is needed on a global scale.     

Alaskan flatfishes on the German market: part 1: identification by DNA and protein analytical methods

North Pacific flatfishes are gaining increased popularity on the German market. Isoelectric focusing of sarcoplasmic proteins and PCR-based DNA analysis were applied to identify fillets of nine Alaskan Flatfish species: Artheresthes stomias (Arrow-tooth flounder), Limanda aspera (Yellowfin sole), Isopsetta isolepis (Butter sole), Lepidopsetta bilineata (Southern rock sole), Lepidopsetta polyxystra (Northern rock sole), Hippoglossus stenolepis (Pacific halibut), Hippoglossoides elassodon (Flathead sole), Platichthys stellatus (Starry flounder), and Glyptocephalus zachirus (Rex sole). Characteristic protein patterns were obtained for raw fillets of several species. Reactivity of flatfish DNA against five pairs of primers was tested, amplifying segments of the mitochondrial cytochrome b, cytochrome oxidase subunit I, 16S rRNA gene, as well as the nuclear parvalbumin gene. Amplicons of the cytochrome b gene were sequenced and used for single-strand conformation polymorphism analysis. The survey of deep-frozen commercial yellowfin sole fillets resulted in the detection of 17% of the fillets being mislabelled; Northern rock sole, butter sole and flathead sole had been used as substitutes.     

搅打稀奶油的乳液特征和打发性能研究

为了对搅打稀奶油的科学应用提供参考,以19款市售代表性搅打稀奶油（常温型、冷藏型和冷冻型产品）为研究对象,通过分析乳液的离心乳析率、黏度、粒径和微观结构研究其乳液的质量,通过分析打发时间、起泡率、泄漏率和裱花性能研究其打发性能。结果显示：常温型产品的离心乳析率为22.17%～32.68%,显著高于冷藏型产品的离心乳析率(1.36%～13.09%)和冷冻型产品的离心乳析率(2.97%～12.87%);常温型和冷藏型产品的黏度、粒径分布特征接近,呈流动性较好且脂肪球分布较均匀的乳液,而冷冻型产品相对黏稠且乳液中无明显脂肪球结构;常温型产品和冷藏型产品的打发时间在13244～291.28 s之间（只有1款冷藏型产品打发时间为79.49 s）,起泡率在111.49%～202.50%之间（只有2款冷藏型产品起泡率分别为92.30%、328.25%）,部分有泡沫泄漏,裱花维持能力较弱;而冷冻型产品打发时间为89.91～158.52 s,起泡率在240.39%～27815%,无泡沫泄漏,裱花维持能力强。综合而言,常温型搅打稀奶油的乳液相对不稳定,打发性能与冷藏型搅打稀奶油接近,而冷冻型搅打稀奶油的打发性能最强。     

Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes

Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10² cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28°C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.     

Comparison and evaluation of molecular methods used for identification and discrimination of lactic acid bacteria

BACKGROUND: Lactobacillus and Bifidobacterium strains are present in a great variety of habitats, including fermented products, probiotic concoctions and the human colon. Some species are so closely related that it is difficult to distinguish them by microbiological techniques. Nevertheless, discrimination of isolates is an important issue in respect of application, and molecular methods such as restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) or species‐specific polymerase chain reaction (PCR) might help in resolving this problem. In this study, PCR, RFLP and sequencing were applied to identify lactobacilli and bifidobacteria originating from various sources and the DSMZ strain collection. RESULTS: The microbiological composition of foods was analysed by molecular methods. Using species‐specific PCR primers, three restriction enzymes (AluI, HhaI and RsaI) and sequencing, three Bifidobacterium and six Lactobacillus reference strains could be distinguished and four additional lactobacilli of food origin were identified. CONCLUSION: A combination of three molecular methods resulted in successful discrimination of nine reference strains and four isolates of food origin. Since these methods are not always accurate owing to their high genetic homogeneity, it is advisable to use more than one method for the identification of L. casei and closely related species. Copyright © 2012 Society of Chemical Industry     

Inhibition of Lipid Oxidation and Rancidity in Precooked Pork Patties by Radical‐Scavenging Licorice (Glycyrrhiza glabra) Extract

This study investigated the efficacy of licorice extract (LE) to curtail lipid oxidation and protect sensory attributes of ground pork during refrigerated and frozen storage. Pork patties (20% fat) were formulated with 0%, 0.02%, 0.05%, and 0.1% (meat basis) LE or rosemary extract (RE) as comparison or 0.01% (fat basis) BHA with 0 or 1.5% NaCl. Raw and precooked (75 °C) patties were packaged in polyvinylchloride overwrapped trays and stored at 2 °C up to 7 and 14 d, respectively, or at –20 °C up to 6 mo. Lipid oxidation (thiobarbituric acid‐reactive substances [TBARS]) and sensory attributes of stored patty samples were evaluated, radical scavenging activity of the LE was measured, and the active phenolic compounds were identified. Cooking yield (<85%) was similar among antioxidant treatments, and lipid oxidation was minimal in refrigerated or frozen raw samples. However, TBARS values in refrigerated precooked control patties (0.22 mg/kg) rose to 9.3 to 9.4 mg/kg after 14 d, compared to 3.4 to 4.4 and 4.4 to 6.9 mg/kg in patties treated with 0.1% LE and RE, respectively. In frozen precooked samples, TBARS (0.22 mg/kg) increased to 1.3 mg/kg (P < 0.05) in control patties after 6 mo and had no significant change in patties treated with 0.1% LE or 0.01% butylated hydroxyanisol. Sensory panel evaluation confirmed strong inhibition of rancidity production by LE, corroborating its remarkable antiradical activity due to the presence of multiple phenolics. The results indicate that licorice has great potential as a natural antioxidative additive to extend the shelf‐life of precooked pork.     

Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication

A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy^® Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.     

Detection of Bacillus cereus on selected retail chicken products

Samples from five chicken meat products, obtained at retail stores, were evaluated for the presence of Bacillus cereus. The products tested were as follows: breaded, fully cooked, frozen nuggets (NUGGETS); breaded, fully cooked, frozen tenders (TENDERS); fully cooked, frozen, white-meat fajita-style strips (STRIPS); raw, refrigerated, boneless, skinless, marinated breast fillets (FILLETS); and raw, refrigerated, cut-up, tray-pack bone-in parts (PARTS), either split breasts or thighs. Four packages of each item were obtained on three different days (n = 60). Frozen and refrigerated products were held overnight in their respective environments as appropriate; then packages were opened aseptically, and a total of 25 g of tissue was excised from multiple pieces within a package. The 25-g samples were enriched in 225 ml of Trypticase soy-polymixin broth for 18 to 24 h at 30 degrees C and then plated on mannitol-egg yolk-polymixin agar and incubated for 18 to 24 h at 30 degrees C. Colonies characteristic of B. cereus were chosen and replated for isolation on mannitol-egg yolk-polymixin agar. Suspect colonies were confirmed as Bacillus spp. by Gram stain, hemolysis on blood agar, and a biochemical test strip. Isolates were further confirmed as B. cereus using Bacteriological Analytical Manual procedures, including tests for motility, rhizoid growth, hemolysis, and protein toxin crystal production. B. cereus was detected in 27 of 60 total samples. By product, the prevalence levels were as follows: NUGGETS, 11 of 12 positive; TENDERS, 8 of 12 positive; STRIPS, 6 of 12 positive; FILLETS, 0 of 12 positive; and PARTS, 2 of 12 positive. Isolates were tested by PCR for presence of the toxin-encoding genes bceT, nheABC, hblACD, and cytK. Results indicate that B. cereus organisms were present on four of the five retail poultry products tested in this study, with the highest rates reported for the three fully cooked items, especially the two breaded products. All strains isolated contained the gene(s) for at least one of the toxins, although none of the strains contained the cytK gene.     

Differentiation of animal species in food by oligonucleotide microarray hybridization

Oligonucleotide microarray hybridization analysis of polymerase chain reaction (PCR) products from the mitochondrial cytochrome b gene DNA was applied to identify different animal species in meat and cheese food samples. A pair of universal primers binding to conserved regions of the vertebrate mitochondrial cytochrome b gene was used to amplify a 377 bp fragment with internal regions of high inter-species variability. PCR products of cattle, pig, chicken, turkey, sheep and goat were unequivocally identified by hybridization with species-specific probe sequences immobilized on an oligonucleotide microarray. In meat samples, 0.1% admixtures of beef or chicken meat were still detectable. By using this new PCR-based DNA chip hybridization for the analysis of 24 commercial food samples from routine control, the simultaneous species composition of mixtures with up to four different species could be determined in a single experiment. The results agreed well with those from the reference methods performed at the local food control authority, which are a combination of enzyme-linked immunosorbent assay (ELISA), species-specific PCR and PCR–RFLP (restriction fragment length polymorphism). Thus, the DNA chip hybridization analysis of cytochrome b PCR products offers a new way for rapid and sensitive species differentiation in food.     

Detection of crustacean DNA and species identification using a PCR-restriction fragment length polymorphism method

The detection of potentially allergenic proteins, such as those derived from crustaceans, in food products is a major concern for the food processing industry. A PCR-restriction fragment length polymorphism (PCR-RFLP) method was designed to detect the presence of crustacean DNA in food products and to determine the species source of the DNA. This PCR assay amplifies an approximately 205-bp fragment of the 16S rRNA gene in crustacean species, including shrimp, crab, lobster, and crawfish. This reaction will not amplify DNA derived from mammals, such as cow and sheep. After amplification, the PCR product is digested with differential restriction endonucleases to determine the species source of the crustacean DNA. The specificity of this assay was demonstrated using four species of shrimp, three species of crab, and two species of lobster and crawfish. This assay is sensitive enough to detect crustacean DNA in a raw meat mixture containing <0.1% shrimp.     

Differentiation of toxigenic Staphylococcus aureusin staphylococcal isolates from prepared and frozen foods by combined arbitrarily primed polymerase chain reaction and DNA probe

In prepared and frozen flamenquín and hake fish fingers Staphylococcus aureus as sanitary hazards have been detected. In the present work, a combined method that includes an arbitrarily primed PCR (AP‐PCR) and a mixed DNA probe hybridisation designed for the enterotoxigenic genes sea, seb, sec, and sed will be assayed to differentiate enterotoxigenic S. aureus from other staphylococcal species isolated during the processing of prepared and frozen foods. From the protocols tested for the AP‐PCR, the highest number of amplification bands showing the best resolution was achieved at 30°C annealing and 35°C extension temperatures. Several staphylococci identified by a biochemical test as S. aureus showed in the AP‐PCR analysis different banding patterns to the references S. aureus. The isolates, were investigated by slot blot hybridisation for genes encoding A, B, C, and D staphylococcal enterotoxins to determine their enterotoxigenic potential. Several isolates characterised by the AP‐PCR analysis as S. aureus hybridised with the DNA probe mixture. The combined AP‐PCR and DNA probe hybridisation assayed was able to differentiate toxigenic S. aureus from other staphylococcal species from prepared and frozen foods. This method could be considered as microbial quality assurance in these products.