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.     

PCR-RFLP authentication of meats from red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus), cattle (Bos taurus), sheep (Ovis aries), and goat (Capra hircus)

PCR-RFLP analysis has been applied to the identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus), cattle (Bos taurus), sheep (Ovis aries), and goat (Capra hircus). PCR amplification was carried out using a set of primers flanking a conserved region of approximately 712 base pairs from the mitochondrial 12S rRNA gene. Restriction site analysis based on sequence data from this DNA fragment permitted the selection of MseI, MboII, BslI, and ApoI endonucleases for species identification. The restriction profiles obtained when amplicons were digested with the chosen enzymes allowed the unequivocal identification of all domestic and game meat species analyzed in the present work.     

Applying DNA techniques to the identification of the species of dressed toasted eel products

To differentiate the species of processed eel products, the gene identification of four fresh eel species was first established and the species of eel products collected from markets were investigated. Polymerase Chain Reaction (PCR) and sequence analysis were used to determine the genetic variation in a 362-nucleotide region of the mitochondrial cytochrome b gene in four fresh eels including Anguilla japonica, Anguilla anguilla, Anguilla rostrata, and Muraenesox cinereus. It was found that each eel species had a unique genotype, which was no different among fresh, frozen, and sterilized meats. The restriction enzyme HinfI could differentiate the species of A. japonica and A. rostrata but could not differentiate A. anguilla and M. cinereus. Another restriction enzyme, Sau96 I, was valuable in the differentiation of M. cinereus from the other three species of Anguilla. By applying PCR and restriction enzymes, the species of 12 commercial eel products were identified as A. japonica (9 samples), A. anguilla (2), and A. rostrata (1). This indicated that the sequence and restriction enzyme cutting site analyses were very usable to authenticate species of different processed eel products.     

Identification of Hake species (Merluccius Genus) using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences.

The use of DNA-based methodologies in identification of hake species belonging to the Merluccius genus was shown to be successful. A short fragment of the left hypervariable domain of the mitochondrial control region was amplified, sequenced, and digested from 11 hake species. The hake-specific PCR product, due to its limited size, was obtained in a variety of tissue samples with different levels of DNA concentration and degradation, including sterilized food products. On the basis of this phylogenetically informative 156-bp sequence were selected four restriction enzymes (ApoI, DdeI, DraIII, and MboII) that allow the hake species discrimination. Species identification by phylogenetic analysis of sequences or by PCR-RFLP methodologies is useful in a variety of scenarios including authentication of thermally processed food, detection of food components, and species determination of individuals whose morphological characters are removed.     

Identification of sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR amplification of the 5S rDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene, has been used for the identification of sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Two species-specific primers were designed to amplify specific fragments of the 5S rDNA gene in each species. The remarkably different size of the amplicons obtained gives, by simple agarose gel electrophoresis, two distinguishable band patterns for both flatfish species. This genetic marker can be very useful for the accurate identification of S. solea and Greenland halibut, to enforce labeling regulations.     

ITS1-rDNA-based methodology to identify world-wide hake species of the Genus Merluccius

Species-specific DNA-based tags are valuable tools for the management of both fisheries and commercial fish products. In this study, we have developed a two-step molecular tool to detect the presence of hake DNA (Merluccius spp.) and to identify the exact hake species present in an blind sample. The first test involves PCR amplification of an ITS1-rDNA fragment of 193 bp using nested primers that are interspecifically conserved in Merluccius spp. and Atlantic cod, Gadus morhua. The second test consists of the PCR amplification of a 602-659 bp DNA fragment spanning part of the ribosomal cluster 18S-ITS1-5.8S and digesting it with four restriction enzymes whose targets map at interspecifically nonconserved sites of the ITS1. Alternatively, the identification of hake species can be achieved by FINS or BLAST, using the nucleotide sequence of either the whole ITS1 sequence or its nested fragment of 193 bp. Because of their high reproducibility and ease of execution, these procedures allow for routine analysis and constitute high reliable tools for the rapid identification of 12 species of hake.     

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).     

黏类小麦CMS不育基因rfv1的分子细胞遗传学跟踪鉴定及定向转育研究

为了实现黏类小麦雄性不育基因rfv1的定向转育,创制优良的黏类非1BL/1RS小麦雄性不育新保持系,本研究以1BS上带有不育基因rfv1的非1BL/1RS小麦变种SP4、莫迦小麦为供体材料,以杀雄剂途径培育的小麦强优势组合西杂1号和西杂5号杂交小麦新品种的母本西农Fp1和西农Fp2为受体材料,采用专一核置换回交转育方法,同时结合根尖细胞学镜检、复合引物PCR及SDS-PAGE和A-PAGE技术,进行不育基因rfv1定向转入的鉴定,旨在育成既携带rfv1不育基因,又具有西农Fp1和西农Fp2核遗传背景的黏类非1BL/1RS小麦雄性不育新保持系。结果如下：（1）根尖体细胞随体鉴定和复合引物PCR分析表明,该法不仅能准确鉴定出1BL/1RS纯合易位系,还可鉴定出1BL/1RS·1BL/1BSrfv1 易位杂合体,两者结果一致。其中复合引物PCR更适合于回交后代中大量目标植株的筛选,为小麦雄性不育基因rfv1定向转移到1BL/1RS易位系提供了准确的鉴定方法与依据;（2）利用SDS-PAGE技术对供试材料进行高低分子量麦谷蛋白亚基的分析表明,在低分子量谷蛋白D亚基区存在莫迦小麦和SP4的特征带;而SP4的高分子量谷蛋白亚基区的6+8亚基,也可以作为示踪小麦雄性不育基因rfv1定向转移到非1BL/1RS易位系的特征亚基条带;（3）A-PAGE技术对醇溶蛋白的分析表明,在ω-醇溶蛋白区也发现莫迦小麦和SP4不同于西农Fp2的特异蛋白条带,也可作为示踪小麦雄性不育基因rfv1定向转移到非1BL/1RS易位系的特征蛋白条带。本研究成功地将小麦杂种优势利用中的杀雄剂法和三系法相结合,促进了小麦杂种优势利用新技术体系的建立。同时该方法亦可应用于黏类小麦雄性不育恢复基因Rfv1的定向转育,进而可实现小麦由生理型不育向遗传型不育的定向转化,以探索一套杂交小麦强优势组合多途径利用的新方法。     

Discrimination of three trachurus species using both mitochondrial- and nuclear-based DNA approaches

A double-DNA approach was developed to discriminate the three Trachurus species that abide in European waters: T. trachurus, T. mediterraneus, and T. picturatus. The analysis aimed at both mitochondrial and nuclear loci. Polymerase Chain Reaction (PCR) amplification of the cytochrome b gene of mtDNA was followed by restriction analysis with three species-specific enzymes: NlaIII, NciI, and BsmAI. Digestion with these endonucleases yielded species-specific electrophoretic profiles. The universality of the results was verified by screening a large number of individuals from 12 geographical regions covering most of the distribution of the species. Additionally, the nuclear multicopy 5S rRNA gene was selected as an alternative candidate for the discrimination of the three Trachurusspecies. A simple agarose gel electrophoretic analysis of the amplicons proved to be capable of leading to unambiguous identification of the three Trachurus species. Thus, the double-DNA methodology presented here allows the accurate discrimination of Trachurus fish species and the detection of commercial fraud.     

PCR-RFLP analysis of nuclear nontranscribed spacer for mackerel species identification

Scomber mackerel have been marketed in fresh and frozen forms and as processed seafood worldwide, and three species of Japanese mackerel S. japonicus, Pacific mackerel S. australasicus, and Atlantic mackerel S. scombrus have constituted a significant part of absolute Scombrid consumption in Japan. The present study was undertaken to develop a rapid and reliable method not only for differentiation of Scomber mackerel from related Scombrid fish by PCR amplification using Scomber genus-specific primers but also for identification of three Scomber mackerel species by PCR-RFLP analysis. Alignment of nucleotide sequences of the nuclear 5S ribosomal RNA gene (5S rDNA) among Scombrid fish allowed the selection of oligonucleotide primers specific for the Scomber genus. These primers enabled amplification of the nontranscribed spacer (NTS) of the 5S rDNA from S. japonicus, S. australasicus, and S. scombrus, whereas no amplification was demonstrated from other Scombrid fish. RFLP analysis of the PCR products with ScaI endonuclease generated unique restriction patterns for each Scomber species. This simple, robust, and reproducible PCR-RFLP technique using Scomber genus-specific primers can serve as a routine food inspection program to enforce labeling regulations of marketed Scombrid fish.     

Validation of a tRNA-Glu-cytochrome b key for the molecular identification of 12 hake species (Merluccius spp.) and Atlantic Cod (Gadus morhua) using PCR-RFLPs, FINS, and BLAST

The goal of this study was to develop a diagnostic key for hake meat to solve the limitations of previous identification methodologies, mainly related to the high degradation of the DNA recovered from processed foods. We describe the development of two molecular tools based on polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphisms of the cytochrome b gene, respectively, to identify DNA from 12 hake species in commercial products. The first assay is an exclusion test consisting of the PCR amplification of a 122 bp fragment using nested primers interspecifically conserved in Merluccius spp. and in Gadus morhua. This 122 bp amplicon, being the shortest one so far designed for hake DNA, is a useful traceability tool for highly degraded samples because its sequence contains enough interspecific diagnostic variation to identify 10 hake species and cod and has been successfully amplified from most commercial products so far tested. The second identification key follows a positive outcome of the exclusion test and consists of the PCR amplification of a 464-465 bp fragment and its digestion with three restriction enzymes whose targets map at interspecifically nonconserved sites of the cytochrome b. The key presented here has passed through a rigorous methodological calibration including its testing for genus specificity, its validation on a large number of authenticated sample types from each species range, and its implementation with a maximum likelihood method for the assignment of unknown samples. Together, these two procedures constitute the most complete molecular key so far developed for Merluccius spp., which is optimal for routine identification of hakes in large commercial samples at a reasonable cost-time ratio.     

Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.     

Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.     

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.     

Fast and efficient discrimination of the Isotoma viridis group (Insecta: Collembola) by PCR-RFLP

Identification of collembolan species is generally based on specific morphological characters, such as chaetotaxy and pigmentation pattern. However, some specimens do not match to described characters because these refer to adult specimens, often of one specific sex, or the characters are highly variable in adults (e.g. pigmentation, setae or furcal teeth). Isozymes have frequently assisted species discrimination, and also these may vary with developmental stage or environmental conditions. For identification of single species of the Isotoma viridis group, we present both direct sequencing of the cytochrome oxidase subunit II (COII) gene and a simple DNA-based molecular method.

Five PCR primers amplifying the COII region (717 bp) of the mitochondrial DNA were used. The sequences clearly separated the species I. viridis, I. riparia and I. anglicana, irrespective of colour varieties within the first species. DNA amplification products of different species can also be distinguished by digestion with restriction endonucleases, followed by gel electrophoresis for separation of fragments. This restriction fragment length polymorphism (RFLP), obtained after digestion with the endonucleases TaqI, VspI, MvaI and Bsp143I, revealed specific fragments that separated the three species from each other. Since restriction enzymes are sensitive to single base mutations, we suggest to use a combination of enzymes with at least two species-specific restriction sites when using the RFLP technique. For the I. viridis complex, VspI and Bsp143I appear to be an appropriate combination.     

苹果基因邻近未知序列的PCR扩增与测序方法

本文提出的方法是首先利用已知基因序列设计三个巢式PCR引物p14、p15和p16,然后设计3’端为常见酶切位点、5’端为随机引物序列p17的9个酶切位点引物,用p14外引物与9个酶切位点引物进行第一轮PCR扩增,其中前5个反应的退火温度较低可以将酶切位点引物的5’端序列引入到PCR产物中,其余反应则以此PCR产物为模板采用较高的正常退火温度进行,退火温度较高是为了使整个酶切位点引物能稳定地结合到最初反应形成的产物上。为提高PCR产物的特异性和产量,用p15和p16引物分别与p17引物配对进行第二轮和第三轮PCR扩增,电泳检查发现PCR产物条带后进行纯化和测序。为保证序列的正确性,利用测得的DNA序列再设计新引物f3x与p14(或p15)配对,直接用提取的DNA为模板进行PCR扩增和测序。利用此方法成功地获得了苹果FPPS基因邻近的启动子序列528bp和5’UTR序列142bp,并已在GenBank注册（登录号FJ263960）。利用Blastn发现这是GenBank中首次发表苹果FPPS基因启动子序列。这说明用该方法获取基因邻近的未知序列是可行的。     

芽孢杆菌TS01的ARDRA评估和遗传分析

利用自主分离的芽孢杆菌菌株TS01和15种芽孢杆菌(地衣芽孢杆菌,枯草芽孢杆菌,短小芽孢杆菌,巨大芽孢杆菌,凝结芽孢杆菌,蜡状芽孢杆菌,迟缓芽孢杆菌,苏云金芽孢杆菌,嗜热脂肪芽孢杆菌,解淀粉芽孢杆菌,环状芽孢杆菌,球形芽孢杆菌,侧孢短芽孢杆菌,多粘类芽孢杆菌,泛酸枝芽孢杆菌)模式菌种进行ARDRA分析。采用16S rDNA通用引物16S-27和16S-1525进行PCR扩增,16S rDNA扩增片段经六种限制性酶（Alu I、Taq I、Mse I、Bst UI、Hha I和Tsp509 I）酶切电泳,获得了TS01菌株的特征性ARDRA指纹图谱。ARDRA图谱通过GelcomparⅡ软件进行聚类分析(UPGMA),结果表明菌株TS01和地衣芽孢杆菌处于同一分支,亲缘关系最近。ARDRA分析鉴定结果与实验室前期菌株TS01形态、生化鉴定和16S rDNA序列分析结果一致,TS01是一株地衣芽孢杆菌菌株,从而证明ARDRA技术在菌种水平上对芽孢杆菌TS01进行鉴别具有可靠性。     

A phylogenetic analysis of the genus Carica L. (Caricaceae) based on restriction fragment length variation in a cpDNA intergenic spacer region

The phylogenetic relationships among twelve wild and cultivated species of Carica (Caricaceae) were analyzed using restriction fragment length variation in a 3.2-kb PCR amplified intergenic spacer region of the chloroplast DNA. A total of 138 fragments representing 137 restriction sites accounting for 5.8% of the amplified region were examined. Both parsimony and neighbor joining cluster analyses confirmed the close association among South American wild Carica species. However, cpDNA data did not support the traditional monophyly hypothesis for the evolution of Carica. Further, cpDNA analyses showed two basic evolutionary lineages within the genus Carica, one defined by cultivated C. papaya and another consisting of the remaining wild species from South America in a well resolved but poorly supported monophyletic assemblage. This evolutionary split in Carica strongly suggests that C. papaya diverged from the rest of the species early in the evolution of the genus and evolved in isolation, probably in Central America.     

Identification of goose,mule duck,chicken, turkey,and swine in foie gras by species-specific polymerase chain reaction

A specific Polymerase Chain Reaction (PCR) has been developed for the identification of goose (Anser anser), mule duck (Anas platyrhynchos x Cairina moschata), chicken (Gallus gallus), turkey (Meleagris gallopavo), and swine (Sus scrofa domesticus) in foie gras. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose, mule duck, chicken, turkey, and swine in foie gras. Analysis of experimental mixtures demonstrated that the detection limit of the assay was approximately 1% for each species analyzed. This genetic marker can be very useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in foie gras.     

Development of a method for the genetic identification of mussel species belonging to Mytilus, Perna, Aulacomya, and other genera

Legislation regarding the labeling of processed products is an important issue in the protection of consumer rights. This labeling is especially important in products that cannot be identified on the basis of their morphological characters, because these are removed from the animal in the transformation process. The goal of this study was the identification of mussel species using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Forensically Informative Nucleotide Sequencing (FINS) methodologies. The molecular marker selected was 18S rDNA (nuclear small-subunit rDNA gene), which allows identification at the genus level and at the species level in some cases. The genera included in this study were Mytilus, Perna, Aulacomya, Semimytilus, Brachidontes, Choromytilus, and Perumytilus. Different markers were used for genetic identification at the species level. To identify the species included in the genus Perna and Choromytilus, a fragment of ITS 1 (Internal Transcribed Spacer 1) was amplified by multiplex PCR and digested with restrictases. The species of Mytilus were identified by length polymorphism and RFLP of the polyphenolic adhesive protein gene. This methodology was validated with products manufactured in the authors' pilot plant and applied to commercial samples. Therefore, this sequential method can be completely or partially used to determine the mussel genus or species present in any food product.