PCR-RFLP analysis of mitochondrial DNA: a reliable method for species identification

A method for identification of game species has been developed on the basis of the amplification of a specific part of the mitochondrial genome (tRNA(Glu)/cytochrome b) using the polymerase chain reaction (PCR). To distinguish between several game species, the obtained 464-bp-long PCR products were cut with different restriction endonucleases (RE) resulting in species-specific restriction fragment length polymorphism (RFLP). Even closely related deer species could be distinguished by application of one or two RE. Natural polymorphisms of the target sequence within one species were examined for red deer (Cervus elaphus), and base pair substitutions were identified affecting the RFLP pattern.     

Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study

Identification of 10 white fish species associated with U.K. food products was achieved using PCR-RFLP of the mitochondrial cytochrome b gene. Use of lab-on-a-chip capillary electrophoresis for end-point analysis enabled accurate sizing of DNA fragments and identification of fish species at a level of 5% (w/w) in a fish admixture. One restriction enzyme, DdeI, allowed discrimination of eight species. When combined with NlaIII and HaeIII, specific profiles for all 10 species were generated. The method was applied to a range of products and subjected to an interlaboratory study carried out by five U.K. food control laboratories. One hundred percent correct identification of single species samples and six of nine admixture samples was achieved by all laboratories. The results indicated that fish species identification could be carried out using a database of PCR-RFLP profiles without the need for reference materials.     

Genetic identification of horse mackerel and related species in seafood products by means of forensically informative nucleotide sequencing methodology

In the present study, a methodology based on the amplification of a fragment of mitochondrial cytochrome b and subsequent phylogenetic analysis (FINS: forensically informative nucleotide sequencing) to genetically identify horse mackerels have been developed. This methodology makes possible the identification of more than 20 species belonging to the families Carangidae, Mullidae, and Scombridae. The main novelty of this work lies in the longest number of different horse mackerel species included and in the applicability of the developed methods to all kinds of processed products that can be found by consumers in markets around the world, including those that have undergone intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 15 commercial samples, all of them canned products. Therefore, these methods are useful for checking the fulfillment of labeling regulations for horse mackerels and horse mackerel products, verifying the correct traceability in commercial trade, and fisheries control.     

PCR technique for identification of mussel species

Random amplified polymorphic DNA (RAPD) analysis has been applied to the identification of four mussels species: Mytilus edulis, Mytilus chilensis, Mytilus galloprovincialis, and Perna canaliculus. Amplifications of DNA from mussel were carried out using random primers. The most distinctive bands were then isolated, cloned, and sequenced to design specific primers. Finally, DNA from different mussels was amplified with these specific primers, and results allow genetic identification of M. galloprovincialis from the rest of the mussel species.     

Evaluation of post-polymerase chain reaction melting temperature analysis for meat species identification in mixed DNA samples

Real-time uniplex and duplex polymerase chain reaction (PCR) assays with a SYBR Green I post-PCR melting curve analysis were evaluated for the identification and quantification of bovine, porcine, horse, and wallaroo DNA in food products. Quantitative values were derived from threshold-cycle (C(t)) data obtained from serial dilutions of purified DNA. The limits of detection in uniplex reactions were 0.04 pg for porcine and wallaroo DNA and 0.4 pg for cattle and horse DNA. Species specificity of the PCR products was tested by the identification of peaks in DNA melting curves, measured as the decrease of SYBR Green I fluorescence at the dissociation temperature. The peaks could be distinguished above the background even at the lowest amount of template DNA detected by the C(t) method. The system was also tested in duplex reactions, by use of either single-species DNA or DNA admixtures containing different shares of two species. The minimum proportions of each DNA species allowing the resolution of T(m) peaks in the duplex reactions were 5% (cattle or wallaroo) in cattle/wallaroo mixtures, 5% porcine and 1% horse in porcine/horse mixtures, 60% porcine and 1% wallaroo in porcine/wallaroo mixtures, and 1% cattle and 5% horse in cattle/horse mixtures. A loss in the sensitivity of the method was observed for some DNA combinations in the duplex assay. In contrast, the results obtained from SYBR Green I uniplex and duplex reactions with single-species DNA were largely comparable to those obtained previously with species-specific TaqMan probes, showing the suitability of that simpler experimental approach for large-scale analytical applications.     

Genetic identification of squids (families Ommastrephidae and Loliginidae) by PCR-RFLP and FINS methodologies

Cephalopods are a taxonomic group that contains a great number of families, genera and species, with many of them very important at the commercial level. The existence of very similar species in this class added up to the transformation process applied to them makes it difficult or even impossible for species identification based on morphological characterization. Moreover, the global commerce makes it possible that one determined species can be marketed in its antipodes. These questions suggest the necessity of molecular techniques to solve this situation. In the present work, a genetic method was developed on the basis of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) technique and makes possible the identification of more than 20 species belonging to the families Ommastrephidae and Loliginidae, as well as some octopus and sepia species. The PCR was employed to amplify 651 and 208 bp fragments of the mitochondrial cytochrome b gene. These molecular systems were applied to fresh, frozen, precooked, even canned cephalopods, allowing for the identification of the species included in these products. Therefore, these molecular tools could be applied in questions related to correct labeling, traceability, and importation controls of squids, sepias, and octopuses.     

Statistical validation of the identification of tuna species: bootstrap analysis of mitochondrial DNA sequences

Sequencing of the mitochondrial cytochrome b gene has been used to differentiate three tuna species: Thunnus albacares (yellowfin tuna), Thunnus obesus (bigeye tuna), and Katsuwonus pelamis (skipjack). A PCR amplified 528 bp fragment from 30 frozen samples and a 171 bp fragment from 26 canned samples of the three species were analyzed to determine the intraspecific variation and the positions with diagnostic value. Polymorphic sites between the species that did not present intraspecific variation were given a diagnostic value. The genetic distance between the sequences was calculated, and a phylogenetic tree was constructed, showing that the sequences belonging to the same species clustered together. The bootstrap test of confidence was used to determine the statistical validation of the species assignation, allowing for the first time a quantification of the certainty of the species assignation. The bootstrap values obtained from these results indicate that the sequencing of the cytochrome b fragments allows a correct species assignation with a probability > or =95%.     

Authentication of coffee by means of PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis

Coffee is one of the most important world food commodities, commercial trade consisting almost entirely of Arabica and Robusta varieties. The former is considered to be of superior quality and thus attracts a premium price. Methods to differentiate these coffee species could prove to be beneficial for the detection of either deliberate or accidental adulteration. This study describes a molecular genetics approach to differentiate Arabica and Robusta coffee beans. This employs a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism to monitor a single nucleotide polymorphism within the chloroplastic genome. Samples were analyzed with a lab-on-a-chip capillary electrophoresis system. Coffee powder mixtures were analyzed with this technique, displaying a 5% limit of detection. The plastid copy number was found to be relatively constant across a wide range of bean samples, suggesting that this methodology can also be employed for the quantification of any adulteration of Arabica with Robusta beans.     

Molecular phylogeny and species identification of sardines

The DNA sequence diversity of Sardina pilchardus (Walbaum, 1792) and some closely related species of Clupeomorpha was investigated using the mitochondrial DNA gene encoding cytochrome b. The nucleotide sequences of complete and partial mtDNA cytochrome b were determined in numerous specimens. Sequence divergence between species and genera was evenly distributed in the cytochrome b gene but rather high compared to reports for other fish species. Phylogenetic analyses on complete cytochrome b were used to study the relationships among the considered species. S. pilchardus was easily differentiated, showing a genetic distance of 0.25 with respect to Clupeidae species and 0.26 with respect to the other species. A species-specific short fragment (<150 bp) was isolated by polymerase chain reaction (PCR) using primers designed for Clupeomorpha. A rapid and reliable PCR method using restriction fragment length polymorphism (RFLP) with two restriction enzymes (MnlI/HinfI) was optimized for unambiguous differentiation of S. pilchardus from the other species tested (raw and canned products).     

Classification of Ilex species based on metabolomic fingerprinting using nuclear magnetic resonance and multivariate data analysis

The metabolomic analysis of 11 Ilex species, I. argentina, I. brasiliensis, I. brevicuspis, I. dumosavar. dumosa, I. dumosa var. guaranina, I. integerrima, I. microdonta, I. paraguariensis var. paraguariensis, I. pseudobuxus, I. taubertiana, and I. theezans, was carried out by NMR spectroscopy and multivariate data analysis. The analysis using principal component analysis and classification of the (1)H NMR spectra showed a clear discrimination of those samples based on the metabolites present in the organic and aqueous fractions. The major metabolites that contribute to the discrimination are arbutin, caffeine, phenylpropanoids, and theobromine. Among those metabolites, arbutin, which has not been reported yet as a constituent of Ilex species, was found to be a biomarker for I. argentina,I. brasiliensis, I. brevicuspis, I. integerrima, I. microdonta, I. pseudobuxus, I. taubertiana, and I. theezans. This reliable method based on the determination of a large number of metabolites makes the chemotaxonomical analysis of Ilex species possible.     

Direct sequencing method for species identification of canned sardine and sardine-type products

A direct sequencing method based on a 103 bp diagnostic sequence derived from a species-specific mitochondrial DNA cytochrome b sequence of 150 bp obtained by Polymerase Chain Reaction was tested for the identification of 47 commercial canned sardine and sardine-type products from various countries. Multiple alignment of 14 analyzed reference samples belonging to Clupeomorpha species was performed versus the canned samples. Low intraspecific variability was observed for canned sardine (相似文献   

U1 and U2 small nuclear RNA genetic linkage: a novel molecular tool for identification of six sole species (Soleidae, Pleuronectiformes)

We evaluated the usefulness of a genetic linkage between the U1 and U2 small nuclear RNAs for species identification. Six soles belonging to the genera Solea, Dicologlossa, and Microchirus were studied. A simple methodology based on two single PCRs is described. Reproducible band profiles were generated for all samples. This rapid and discriminatory molecular method is highly promising for determining the authenticity of sole fillets in the food industry.     

Comparative analysis of organelle DNAs in acid citrus grown in Japan using PCR-RFLP method

PCR-RFLP analyses of three regions for each of chloroplast DNA (cpDNA; rbcL-ORF106, trnD-trnT, trnH-trnK) and mitochondrial DNA (mtDNA; nad7/exon2-exon3, nad7/exon3-exon4, 18S-5S) were performed in 26 cultivars of acid citrus grown in Japan to identify polymorphisms and classify them. The polymorphisms were compared with those of three true Citrus species, i.e., mandarin, pummelo and citron. Ichang papeda (C. ichangensis) was also included in this study to find its relationship with Yuzu. Inter-species cpDNA variation was recognized and the acid citrus were divided into three groups, namely; I (‘Yuzukichi’ and ‘Kinkoyu’), II [sour oranges (‘Kaiseito’, ‘Daidai’ and ‘China daidai’), ‘Nansho daidai’, ‘Kiku daidai’, C. sudachi (‘Mushi yukaku’, ‘Yushi yukaku’ and ‘Yushi mukaku’), C. sphaerocarpa (‘Kabosu’ and ‘Aka kabosu’), C. kizu (‘Taninaka kizu’, ‘Kinosu’ and ‘Kizu’), ‘Zanbo’, ‘Mochiyu’, ‘Jabara’ and ‘Naoshichi’], and III [Yuzu (‘Tetraploid’, ‘Tochikei yuzu’ and ‘Yamanekei yuzu’), ‘Matsuda sudachi’, ‘Zuishoyu’, ‘Hanayu’ and ‘Yuko’]. CpDNA restriction patterns of the three true Citrus species differed from each other as well as from those of ichang papeda. CpDNA restriction patterns of group I of the acid citrus were identical to those of mandarins. Group II showed the same as pummelos. CpDNA restriction patterns of group III were differed from those of the three true Citrus species in the three regions. This group was differed from ichang papeda after digestion of trnH-trnK PCR products with TaqI, HinfI and AluI, while they showed identical restriction patterns in two regions, rbcL-ORF106 and trnD-trnT. Citrons and ichang papeda were placed in groups IV and V, respectively. Based on mtDNA restriction patterns, the acid citrus were divided into three groups; i, ii and iii. In groups i and ii accessions of groups I and II of cpDNA were placed with mandarins and pummelos, respectively. In group iii accessions of group III of cpDNA were placed with ichang papeda. Citrons were placed in a distinct group, iv.     

Diversity and evolution of major Panax species revealed by scanning the entire chloroplast intergenic spacer sequences

Ginseng is an important medicinal plant, but almost no genomic information is known for it. For the primary step to understand the Panax genome, we inspected the chloroplast genome sequence diversity and used that to infer the evolution of Panax species using them. We inspected a total of 101 intergenic spacers (IGS) covering 44,563 bp (96.8 % of the total IGS) from four Panax species. Diversity was inspected by three steps: gel electrophoresis, high resolution melting (HRM) analysis, and further confirmation by sequencing. Even though low levels of InDel polymorphism were detected by gel electrophoresis, high levels of reproducible polymorphisms were identified by HRM analysis. Sequencing of the HRM-polymorphic spaces and pair-wise multiple sequence alignments revealed up to 247 sequence variations between species. We have identified 62 IGS showing polymorphism between species. Among them, the trnE-trnT, trnT-psbD, ndhF-rpl32, and rpl14-rpl16 spaces are more informative for studying the diversity of Panax relatives. Phylogenetic analysis and molecular evolution studies revealed that P. notoginseng is most diverged from the other Panax species, with a nucleotide substitution rate of 0.0039. P. quinquefolius shows a close relationship with P. ginseng and P. japonicus, with a nucleotide substitution rate of 0.0009. Meanwhile, no sequence variation was detected between P. ginseng and P. japonicus. Calculation of molecular clocks revealed that P. notoginseng diverged more than 1.30 million years ago from the other Panax species and then, P. quinquefolius diverged from P. ginseng by migration in the American continent more than 0.29 million years ago.     

Isozyme analysis of indigenous cassava germplasm for identification of duplicates

Isozyme analysis was utilized for identification of duplicates in indigenous collection of cassava germplasm. 786 indigenous accessions of cassava were screened for 11 key morphological characters and the morphological duplicates were identified. Two hundred and eighteen accessions of cassava consisting of ninety one sets of morphological duplicates were analysed for esterase isozyme polymorphism. Thirty seven polymorphic bands were obtained, showing high polymorphism for this enzyme. Thirty seven double sets and 17 multiple sets were found to be similar at isozyme level. These isozyme duplicates will be further analysed for DNA similarity before eliminating them from the field germplasm. The dissimilar sets showed variation in minor morphological characters, thus confirming the relation between isozyme polymorphism and phenotypic variation.     

Quantitative bioluminometric method for DNA-based species/varietal identification in food authenticity assessment

A method is reported for species quantification by exploiting single-nucleotide polymorphisms (SNPs). These single-base changes in DNA are particularly useful because they enable discrimination of closely related species and/or varieties. As a model, quantitative authentication studies were performed on coffee. These involved the determination of the percentage of Arabica and Robusta species based on a SNP in the chloroplastic trnL(UAA)-trnF(GAA) intraspacer region. Following polymerase chain reaction (PCR), the Robusta-specific and Arabica-specific fragments were subjected to 15 min extension reactions by DNA polymerase using species-specific primers carrying oligo(dA) tags. Biotin was incorporated into the extended strands. The products were captured in streptavidin-coated microtiter wells and quantified by using oligo(dT)-conjugated photoprotein aequorin. Aequorin was measured within 3 s via its characteristic flash-type bioluminescent reaction that was triggered by the addition of Ca(2+). Because of the close resemblance between the two DNA fragments, during PCR one species serves as an internal standard for the other. The percentage of the total luminescence signal obtained from a certain species was linearly related to the percent content of the sample with respect to this species. The method is accurate and reproducible. The microtiter well-based assay configuration allows high sample throughput and facilitates greatly the automation.     

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.     

Structural identification of nonvolatile dimerization products of glucosamine by gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and nuclear magnetic resonance analysis

The degradation profile of glucosamine bulk form stressed at 100 degrees C for 2 h in an aqueous solution was studied. Column chromatography of acetylated product mixture led to isolation of two pure compounds (1b and 2b) and a mixture of at least three isomers (3b). 1a and 2a were identified as 5-(hydroxymethyl)-2-furaldehyde (5-HMF) and 2-(tetrahydroxybutyl)-5-(3',4'-dihydroxy-1'-trans-butenyl)pyrazine, respectively, by utilizing a variety of analytical techniques, such as GC-MS, LC-MS, on-line UV spectrum, (1)H and (13)C NMR, and DEPT, as well as (1)H-(1)H COSY. 3a was identified as 2-(tetrahydroxybutyl)-5-(2',3',4'-trihydroxybutyl)pyrazine, commonly known as deoxyfructosazine. In addition, glucosamine solid dosage form was exposed to 40 degrees C/75% relative humility for 10 weeks. Methanol extract of glucosamine solid dosage form was analyzed after acetylation by LC-MS, resulting in degradants 3b and 4b. 3a and 4a were, therefore, determined as deoxyfructosazine and 2,5-bis(tetrahydroxybutyl)pyrazine (fructosazine), respectively. Furthermore, the mechanisms of formation of identified degradation products are proposed and briefly discussed.     

Evidence of rapid evolution and incipient speciation in Vicia sativa species complex based on nuclear and organellar RFLPs and PCR analysis

Seventy three accessions of the sevenVicia species belonging to Sativa speciescomplex were screened for nuclear and organellar restriction fragmentlength polymorphic (RFLP), and random amplified polymorphicDNA (RAPD) markers. Total genomic DNAs of 73 accessions wasrestricted with three enzymes, and the restriction fragments werehybridized to the wheat rDNA probe pTa71 (containing 18S, 5.8Sand 25S rDNA genes, and spacers), and faba bean probes Ver6-5 (entire intergenic spacer flanked by small part of25S and 18S fragments) and Ver 18-6 (part of thecoding region of the gene and internal transcribed spacers). InXbaI digests, 16 repeat unit length classes in24 combinations were identified. Digestion withEcoRI and DraI gave2–4 and 1–3 fragments, respectively, with detectablehybridization to the probes, indicating the existence of internalXbaI sites. All the accessions produced 3.5 EcoRI fragment arising fromcoding region of the repeat unit. Four hundred and eighteen RAPDmarkers among 45 accessions were identified with 14 arbitrary10-base primers. The percentage of polymorphic bands withinspecies ranged from 20 in V.angustifolia to 98% inV. nigra. Both RFLP andRAPD markers were unable to assess the relationships betweenaccessions within species as there was often much closer resemblancesbetween certain accessions of different species rather than betweenaccessions within each taxon. This analysis supports the view basedon morphological, cytogenetical and crossability data that it is notpossible to classify Sativa species complex into a small finitenumber of taxa which are clearly circumscribed, and that the complexrepresents a unique case of rapid evolution and incipient speciation.A study of chloroplast and mitochondrial RFLPs was undertaken toanalyze phylogeny through maternal lineage. Chloroplast DNArestriction fragment patterns, using 13 restriction endonucleases,revealed 92.6 to 99% homology between the seven species.Twelve enzyme-probe combinations yielded identical fragmentpatterns for all the seven species. The molecular sizes of thechloroplast DNAs obtained were similar (121.5–123.5), indicating that they had all lost one of theinverted repeats. Total DNAs digested with three restriction enzymesand hybridized to six heterologous probes of mitochondrial originyielded monomorphic bands in five enzyme—probe combinationsacross all the 73 accessions. In other combinations as well,40–66 accessions yielded monomorphic profiles. The smallvariation in the remaining accessions was not species-specificsince the same profiles were present in more than one species. Theseresults i) strongly suggest that the seven species within thecomplex share a common ancestor, or direct lineage and, ii)indicate that these species should be relegated to a rank, perhaps ofsubspecies, within V.sativa species complex.     

Development of a method for the genetic identification of flatfish species on the basis of mitochondrial DNA sequences

In the present study a method for genetic identification of flatfish species was developed. The technique is based on DNA sequencing of amplified DNA by PCR and subsequent phylogenetic analysis ( FINS). A phylogenetic tree using the cytochrome oxidase subunit I (COI) was constructed and the bootstrap values calculated. The mentioned technique allows the genetic identification of more than 50 flatfish species in fresh, frozen, and precooked products. This analytical system was validated and subsequently applied to 30 commercial samples, obtaining 13 that were incorrectly labeled (43%). Four of the mislabeled samples were whole fish (31%), and nine were fillets (69%). The species with the higher rate of incorrect labeling were Pleuronectes platessa (17%) and Solea solea (10%). Other species incorrectly labeled were Hipoglossus hipoglossus (7%), Reinharditus hippoglossoides, Limanda ferruginea, and Microstomus kitt (3% each species). Therefore, this molecular tool is appropriate to clarify questions related with the correct labeling of commercial products, the traceability of raw materials, and the control of imported flatfish, and also can be applied to questions linked to the control of fisheries.