Molecular identification of nine commercial flaffish species by polymerase chain reaction-restriction fragment length polymorphism analysis of a segment of the cytochrome b region

Commercial refrigerated or frozen flatfish fillets are sometimes mislabeled, and identification of these mislabeled products is necessary to prevent fraudulent substitution. Identification of nine commercial flatfish species (order Pleuronectiformes), Hippoglossus hippoglossus (halibut), Lepidorhombus boscii (four-spotted scaldfish), Lepidorhombus whiffiagonis (megrin), Platichthys flesus (flounder), Pleuronectes platessa (European plaice), Reinhardtius hippoglossoides (Greenland halibut), Scophthalmus maximus (turbot), Scophthalmus rhombus (brill), and Solea vulgaris (=Solea solea) (sole), was carried out on the basis of the amplification of a 486-bp segment of the mitochondrial genome (tRNA(Glu)/cytochrome b) by using the polymerase chain reaction (PCR) and universal primers. Sequences of PCR-amplified DNA from the flatfish species were used to select eight restriction enzymes (REs). The PCR products were cut with each RE, resulting in species-specific restriction fragment length polymorphism. Seven species groups could be identified by application of the single RE DdeI and six species groups by using HaeIII, HinfI, MaeI, or MboI. Different combinations of only a couple of these REs could unambiguously identify the nine flatfish species. Genetic polymorphisms of the target sequence were examined by comparison with previously published DNA sequences, and the results of this comparison confirmed the usefulness of this technique in distinguishing and genetically characterizing refrigerated or frozen pieces of these nine flatfish species.     

PCR-RFLP analysis of the rpoB gene to distinguish the five species of Cronobacter

Members of the genus Cronobacter are opportunistic pathogens associated with life-threatening infections in immuno-compromised individuals. Polyphasic analysis has facilitated the classification of the novel genus Cronobacter containing five species. However, since this recent reclassification there are not many identification methods optimised for differentiation between the five Cronobacter species. This differentiation between the species is of importance as there are indications that the species may be diverse regarding their virulence. The aim of this study was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol to differentiate between the five Cronobacter species. The rpoB gene of 49 Enterobacteriaceae strains, including 33 Cronobacter strains was amplified using conventional PCR, followed by digestion of these PCR products with restriction endonucleases MboI, HinP1I and Csp6I. The PCR-RFLP analysis with single digestions of each of the restriction endonucleases did not distinguish between all five Cronobacter species. This study describes the successful differentiation of the five Cronobacter species based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. This PCR-RFLP assay is an accurate identification method that ensures rapid differentiation between the five species of Cronobacter.     

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     

Development of a Polymerase Chain Reaction System for the Detection of Dog and Cat Meat in Meat Mixtures and Animal Feed

The identification of species origin of meat represents a considerable problem for food and animal feed analysis. In the present study a PCR‐mediated method for the detection of dog and cat meat was developed. For this the cytochrome b gene sequence of both species was analyzed by restriction fragment length polymorphism (RFLP) analysis. The use of the restriction enzymes Alu I and Hae III yielded specific restriction profiles characteristic for each species. The meat of both species could additionally be differentiated with species‐specific oligonucleotide primers based on specific parts of the cytochrome b gene sequences characteristic for dog and cat. The use of these oligonucleotide primers allowed a direct identification of dog and cat meat in meat mixtures even after heat treatment.     

Identification of gadoid fish species using DNA-based techniques

The polymerase chain reaction (PCR) technique was employed to obtain a 464 bp amplicon from the mitochondrial cytochrome b gene from gadoid species to study its ability to differentiate them. The sequences of this fragment from 16 species were analysed using a genetic distance method, and polymorphic sites were determined. The fragment was shown to be moderately polymorphic (151 sites), and this permitted the differentiation of most of the species. A phylogenetic tree construction using Tamura-Nei distances was employed to allow the identification of Gadidae species, each species resulted in a well-differentiated clade, with the exception of Gadus ogac and Gadus macrocephalus, which could not be differentiated. Based on the sequences obtained, three restriction enzymes, Dde I, Hinc II and Nla III, were selected to provide specific restriction profiles, which allowed the differentiation of 15 species of gadoids in a faster and less expensive way than sequencing. The PCR-restriction fragment length polymorphism methodology was also tested using commercial samples.     

Identification of gadoid species (Pisces, Gadidae) by sequencing and PCR–RFLP analysis of mitochondrial 12S and 16S rRNA gene fragments

We applied PCR–RFLP (Polymerase Chain Reaction – Restriction Fragment Length Polymorphism) analysis to identify seven gadoid species of different biogeographical origin and commercial relevance, namely Gadus morhua (Atlantic ocean); Trisopterus minutus capelanus, Trisopterus minutus minutus, Molva elongata, Phycis blennoides, Micromesistius poutassou (Atlantic ocean and Mediterranean sea); Theragra chalcogramma (Pacific ocean). Two DNA fragments belonging to mitochondrial 12S and 16S rRNA genes of about 430 and 630 bp, respectively, were isolated by PCR amplification. Their direct sequencing showed a significant genotypic diversity among gadoid species, useful for species identification. Digestion of 16S rRNA gene PCR fragment with MvaI or Bsh1285I restriction enzymes, followed by agarose gel electrophoresis of the cleaved products, yielded specific restriction profiles that enabled direct, visual identification of the species analyzed. This PCR–RFLP method allowed a clear and rapid discrimination of the gadoid species studied.     

Duplex real-time PCR for authentication of anglerfish species

A duplex real-time PCR assay based on TaqMan probe technology was developed for the authentication of two commercially important anglerfish species (Lophius budegassa and L. piscatorius). This technique uses the cytochrome oxidase subunit I and allows the unambiguous identification of this species. It is notable for its simplicity, rapidity, highest potential for automation and minor risk of contamination. The TaqMan real-time PCR is currently the most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of these species. The method can be applied to all kind of products as fresh, frozen, and precooked products. The developed methodology, which uses two specific primer–probe sets, has been validated and subsequently applied to 40 commercial samples labeled as any anglerfish species in order to determinate whether the species used for their manufacturing correspond to these species. The methodology herein developed is appropriate to clarify questions related with the correct labeling of commercial products, traceability in commercial trade, and fisheries control.     

Survey of the authenticity of prawn and shrimp species in commercial food products by PCR-RFLP analysis of a 16S rRNA/tRNA mitochondrial region

A novel PCR-RFLP method was evaluated as a tool to assess the incidence of incorrect labelling of prawns and shrimps in commercial food products. The whole method can be performed in less than 8 h in only one day of work. PCR amplification with primers 16Scru4/16Scru3, targeted to the amplification of a ca. 530 bp region of 16S rRNA and tRNA^Val mitochondrial genes, was coupled to restriction analysis with AluI, TaqI or HinfI. Forty-one commercial food products were considered. The molecular method considered allowed the identification of up to 17 different prawn and shrimp species in all the processed products considered. Seven (28%) of the 25 food products declaring one or more species in their labels were incorrectly labelled. Authentication was successfully assessed in commercial peeled products subjected to industrial processing, in which none of the products displayed labelling at species level. Overall, incorrect labelling was detected in 10 (24.4%) of the 41 commercial products tested, while another 16 samples (39%) exhibited incomplete labelling. The molecular method evaluated in this study proved to be a rapid and easy-to-perform two-step analytical approach to achieve species identification of commercial whole specimens of frozen prawns and shrimps and in peeled processed products where such raw materials are included as added-value ingredients.     

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     

Genetic Identification of Lophius budegassa and L. piscatorius by PCR-RFLP Analysis of a Mitochondrial tRNAGlu/Cytochrome bSegment

Identification of Lophius budegassa(black‐bellied angler) and L. piscatorius(angler) (Lophiiformes) was carried out on the amplification of a 486 bp tRNA^Glu/cytochrome b segment using the polymerase chain reaction (PCR). Direct DNA sequencing of 6 PCR products was carried out. Six restriction endonucleases (AluI, CfoI, HaeIII, HinfI, Mae, and ScrFI) with different species‐specific restriction fragment length polymorphism (RFLP) were selected. Digestions of PCR products from 30 individuals showed no intraspecific polymorphism. Double digestions (CfoI and HinfI, and HaeIII and ScrFI) were simpler and more rapid than single digestions. This technique is suitable for distinguishing tails of both Lophius species.     

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     

Differentiation of lactic acid bacteria based on RFLP analysis of the tuf gene

The tuf gene, which encodes the elongation factor Tu, is universally distributed in Gram-positive bacteria. A small region of the tuf gene was evaluated for the differentiation of Lactobacillus strains using restriction fragment length polymorphism (RFLP) analysis. The 803 bp portion of tuf genes from 9 closely-related Lactobacillus reference strains were successfully amplified by polymerase chain reaction (PCR), and restriction patterns of each were evaluated with a set of restriction enzymes, which included AluI, HaeIII, and Sau3AI. The results implied that restriction patterns obtained from 2 of these restriction enzymes (AluI and HaeIII) could effectively differentiate closely-related Lactobacillus species.     

Microbiological evaluation and molecular characterization of bifidobacteria strains in commercial fermented milks

The aim of this study was to evaluate the presence of Bifidobacterium animalis subsp. lactis in commercial dairy products using different molecular techniques. We analyzed the microbiological composition of 13 commercial fermented milks available in the Spanish market. Thirteen strains of genus Bifidobacterium were isolated from these products and were identified by genus-specific PCR, by fluorescence in situ hybridization (FISH), by multiplex PCR and amplified ribosomal DNA restriction analysis (ARDRA). The same sets of strains were typed by randomly amplified polymorphic DNA (RAPD) analysis and by amplified fragment length polymorphism technique (AFLP). All strains were identified as B. animalis subsp. lactis using ARDRA and multiplex PCR techniques. Similarity between strains was evaluated based on RAPD and AFLP profiles. The isolated strains showed similar profiles by using these techniques, revealing the reduced genetic variability existing among commercial strains, and all these profiles were reproducible in repeated analysis. ARDRA and multiplex PCR are techniques that allow differentiation of the bifidobacteria at genus and species level, but do not indicate if they are different strains, for which reason the RAPD technique is very useful. All bifidobacteria isolated from commercial fermented milks in Spain belong to the same species B. animalis subsp. lactis. Our results demonstrate the necessity to control the presence of bifidobacteria in commercial fermented milks, not only at species level but also at strain level. Multiplex PCR and RAPDs are the most suitable, rapid and precise techniques to identify all bifidobacteria contained in fermented milk products at genus-, species-, and strain levels.     

Meat species identification and Halal authentication analysis using mitochondrial DNA

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.     

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.     

Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait

BACKGROUND: Dried root of Sophora flavescens Ait. is a medicinal material occasionally misused or adulterated by other species similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)‐generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. RESULTS: The S. flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR‐RFLP coupled with restriction enzymes Sac I, Sac II, Xho I or Pvu I produced specific fragments for all tested variants. ITS2 PCR‐RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S. flavescens Ait. and Sophora tomentosa L. in various ratios. The developed ITS2 PCR‐RFLP markers could detect mixed DNA samples of S. flavescens Ait./S. tomentosa L. up to a ratio of 10:1. CONCLUSION: The present study demonstrates the usefulness of ITS2 PCR‐RFLP coupled with pre‐selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants. Copyright © 2011 Society of Chemical Industry     

A rapid PCR–RFLP method for the identification of Lophius species

The seven Anglerfish species, which belong to the genus Lophius, have a different value on the market, worldwide. If whole fishes can be identified by their morphological characteristics, they become indistinguishable when prepared or processed. In this study, a rapid method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was developed for the authentication of the seven Lophius species, using a cytochrome b gene fragment of 566 bp. After a genus-specific PCR, a fast digestion with the restriction enzyme BfaI, followed by agarose gel electrophoresis, allowed a clear species identification by producing specific restriction patterns. The total time required was as low as 6 h, DNA extraction included. The method was then used to analyse 48 commercial samples, whose phylogenetic analysis confirmed the PCR–RFLP response at 100 %. Results showed that mislabelling occurs on the market regardless the kind of processing.     

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.     

Survey of authenticity of meat species in food products subjected to different technological processes,by means of PCR-RFLP analysis

Polymerase chain reaction (PCR) coupled to restriction-fragment length polymorphism analysis (RFLP) was considered for exploring the incidence of incorrect labelling in food products containing one or more meat species. Universal primers CYT b1/CYT b2, which amplify a variable region of the mitochondrial cytochrome b of vertebrates, and endonucleases PalI, MboI, HinfI and AluI were used for this purpose. Fifty food products, nine of them raw or cured and the other 41 subjected to a variety of technological processes such as pre-cooking and freezing, cooking and smoking, dehydration or sterilisation, were investigated. Twenty of the 50 products declared mixtures of meat species on their labels. Fifteen (30%) of the 50 food samples investigated displayed an incorrect qualitative labelling. While this affected only one (11.1%) of the nine raw/cured products, 14 (34.2%) of the 41 products subjected to some type of heat-processing were not correctly labelled. The undeclared presence of turkey was the most frequent concern, since it was detected in seven food products. The complete absence of a declared species of high commercial value—such as beef or roe-deer—was observed in another four cases. The PCR-RFLP method used here proved to be a rapid and easy-to-perform two-step analytical approach to achieve qualitative meat species identification in raw and cooked food products containing one or more different species.