Genetic differentiation between the clam species Ruditapes decussatus (grooved carpet shell) and Venerupis pullastra (pullet carpet shell) by PCR–SSCP analysis

Genetic differentiation of the clam species Ruditapes decussatus (grooved carpet shell) and Venerupis pullastra (pullet carpet shell) has been achieved based on polymerase chain reaction–single‐strand conformation polymorphism (PCR–SSCP) analysis. A short fragment (150 bp) of the α‐actin gene was amplified by PCR. Amplicons were denatured to obtain single‐stranded DNA, electrophoresed on a non‐denaturing polyacrylamide gel and visualised by silver staining for detection of SSCPs. Species‐specific DNA band patterns were obtained for R decussatus and V pullastra, allowing clear differentiation of the two clam species. © 2002 Society of Chemical Industry     

Polymerase chain reaction-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment for authentication of four clam species

Specific identification of four clam species, Ruditapes decussatus (grooved carpet shell), Venerupis pullastra (pullet carpet shell), Ruditapes philippinarum (Japanese carpet shell), and Venerupis rhomboides (yellow carpet shell), was achieved by polymerase chain reaction-restriction fragment length polymorphism analysis of a fragment of the mitochondrial 16S rRNA gene. Amplification of DNA isolated from the foot muscle produced fragments of 511 bp for V. pullastra, 523 bp for R. decussatus, 545 bp for R. philippinarum, and 502 bp for V. rhomboides. The restriction profiles obtained by agarose gel electrophoresis when amplicons were digested with endonucleases BsmAI and BsrI allowed unequivocal identification of the four clam species. This approach would be less costly, simpler, and quicker than conventional sequencing of polymerase chain reaction products followed by detailed comparison of individual sequences, especially when large numbers of samples need to be analyzed.     

Identification of European commercial cockles (<Emphasis Type="Italic">Cerastoderma edule</Emphasis> and <Emphasis Type="Italic">C. glaucum</Emphasis>) by species-specific PCR amplification of the ribosomal DNA ITS region

We present a novel one-step PCR method for the identification of Cerastoderma edule and C. glaucum. Sequence differences found in the internal transcribed spacer region (ITS) of the ribosomal DNA of the two cockles allowed us to design two species-specific reverse primers. These two primers were used in a multiplex reaction, together with a forward universal primer to amplify specific fragments with different lengths in each cockle (190 and 470 bp in C. edule and C. glaucum, respectively). The successful and specific amplifications obtained for two natural populations in each species as well as in canned products lend support to the usefulness of these markers.     

Effect of warming on protein,glycogen and fatty acid content of native and invasive clams

Human bivalve consumption in Europe has steadily increased in the last years, particularly during summer months when seawater temperature increases. Since ocean warming is among the current global environmental threats affecting aquatic organisms, it is of paramount importance to investigate its effect on the nutritional quality of seafood products. In this context, the aim of this study was to investigate differences in the nutritional quality (in terms of protein, glycogen and fatty acid, FA, content) and condition of a native (grooved carpet shell, Ruditapes decussatus) and an invasive (Japanese carpet shell, Ruditapes philippinarum) clam species, subjected to warming. Our results clearly reveal that temperature significantly affected the nutritional quality of both clam species, particularly the FA composition. Both clam species responded similarly to warming, by significantly decreasing the content of some fatty acids, but not protein and glycogen levels. A predominance of polyunsaturated FA (PUFA) over saturated FA (SFA) and monounsaturated FA (MUFA) was observed throughout the experiment, as well as high n − 3/n − 6 and PUFA/SFA ratios. The native clam always revealed higher values of these fatty acids, indicating that this species has a better nutritional quality in comparison to the invasive one. Nonetheless, the loss of n − 3 PUFA (in native species), eicosapentaenoic (EPA; in both species) and docosahexaenoic (DHA; in invasive species) acids was considered as the major negative outcome derived from warming, since it contributes to the loss of prime quality fatty acids for human health. However, atherogenic, thrombogenic and hypocholesterolemic/hypercholesterolemic indices (AI, TI and h/H, respectively) remained low in both species, even in warming conditions, suggesting that these food items can be used in a cardio-protective and hypocholesterolemic diet. This study provides new insights to understand and foretell the effects of climate change on nutritional quality of marine organisms.     

Development of a Novel PCR Primer to Differentiate and Identify Bacillus subtilis and Closely Related Species Isolated from Thai Fermented Foods

A novel PCR assay based on 16S-23S internal transcribed spacers (ITS) length polymorphism was developed for rapid differentiation and identification of the Bacillus subtilis group, especially B. subtilis, B. licheniformis and B. amyloliquefaciens, the most frequently isolated bacilli from fermented foods. A new group-specific conserved primer pair, CITS-F and CITS-R, was designed for specific amplification of the ITS region in B. subtilis and closely related species. The fingerprints of seven reference species, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. atrophaeus, B. vallismortis and B. mojavensis using CITS-F and CITS-R primers showed the same four signature bands of 227, 400, 542 and 650 bp. They are different from those of other genera and species tested. Therefore, these four signature bands could be used to differentiate and identify the B. subtilis group. Moreover, the sequence of the 227 bp signature band could also be used to distinguish closely related species of the B. subtilis group used in this study. A novel PCR assay based on ITS length polymorphism pattern using CITS-F and CITS-R could be considered as a rapid and easy method for the primary differentiation of the B. subtilis group.     

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.     

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     

Molecular approaches for the detection of truffle species in processed food products

Molecular identification techniques were applied in order to analyse food products containing fragments of some Tuber species. Samples of fungal DNA were processed by analyses of the internal transcribed spacer (ITS) region. The polymerase chain reaction (PCR) using truffle species‐specific primers, multiplex PCR, restriction fragment length polymorphism (RFLP) analysis, sequencing of the ITS region and specific oligonucleotide probe hybridisation were used. The results obtained demonstrate the applicability of these molecular strategies to the identification of truffles, even when their morphological characteristics are difficult to interpret owing to the drastic treatments utilised in food preparation or the use of unripe fruit bodies (lacking spores). Furthermore, testing was also possible starting from very small amounts of sample and degraded DNA. The methods described have important applications in both the production and sale of such food products, in order to avoid fraud and reveal the possible presence of other fungal species. © 2002 Society of Chemical Industry     

Combination of SYBR Green II and TaqMan Probe in the adulteration detection of Dendrobium devonianum by fluorescent quantitative PCR

‘Zipi Fengdou’ is a restorative food made of the stem of Dendrobium devonianum, Paxt., whose high commercial value presents the constant risk of adulteration with cheaper ‘Foudou’ products made from other Dendrobium species. Therefore, this study developed two assays capable of detecting D. devonianum based on SYBR Green II and TaqMan probe real‐time PCR. By performing the diagnostic real‐time PCR based on SYBR Green II, two DNA fragments (163 and 159 bp) were specifically amplified from the internal transcribed spacer (ITS) region of D. devonianum. The average cycle threshold (C_t) values of two D. devonianum samples collected from different Chinese provinces were shown to be 16.65 for ITS1‐4 and 16.38 for ITS2‐1, which were significantly (P < 0.001, SPSS) different from those of the reference Dendrobium species used as adulterants of ‘Zipi Fengdou’ (34.94 for ITS1‐4, 34.67 for ITS2‐1). Moreover, in the assay based on TaqMan probe real‐time PCR, two TaqMan probes were designed and tested for the quantitative detection of D. devonianum in commercial samples labelled as ‘Zipi Fengdou’. As a result, this assay specifically and sensitively distinguished the processed Fengdou’ sample of D. devonianum from those of adulterant Dendrobium species.     

Specific identification of Candida albicans by hybridization with oligonucleotides derived from ribosomal DNA internal spacers

In order to develop DNA probes for rapid, sensitive and specific detection of the pathogenic yeast species Candida albicans, we carried out comparative sequence analysis of the two internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA (rDNA) units of C. albicans and the closely related pathogenic species C. tropicalis. While overall sequence similarity between the two species was considerable (65–75%), both ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific target sites for the identification of C. albicans. On the basis of these results one ITS1-derived ( ANAB1 ) and two ITS2-derived ( ANAB2 and ANAB3 ) oligonucleotides were selected, chemically synthesized, and used as hybridization probes. Their specificity and reliability were evaluated in dot-blot hybridization experiments with total genomic DNA from 13 strains of medically important Candida species, six strains of other yeast genera associated with man and animals, and ten strains previously identified as C. albicans by phenotypic criteria. Under well-defined hybridization conditions the three probes hybridized exclusively with DNA derived from strains belonging to the species C. albicans, thus demonstrating their potential clinical usefulness. The failure of four of the (presumed) C. albicans strains to show hybridization to the ITS probes sheds doubt upon their taxonomic classification, which is reinforced by other phenotypic aspects of these strains.     

用ITS2鉴定芥辣类调味品中山葵、辣根和芥末组分

日式饮食中的芥辣类调味品主要以同属十字花科的山葵(Eutrema wasabi)、辣根(Armoracia rusticana)、芥末(mustard)做原料加工而成。利用核糖体DNA内部转录间隔区Ⅱ(Internal transcribed spacer 2,ITS2)技术快速、准确地鉴定出山葵、芥末和辣根,为该技术应用于芥辣类产品质量检测提供参考。以3种植物材料(山葵、辣根、白芥末)为实验材料,提取基因组DNA,通过引物ITS2_S2进行PCR扩增得到ITS2片段,测序结果通过生物信息学分析进行物种鉴定。MEGA7.0(Molecular evolutionary genetics analysis)分析ITS2序列结果表明,山葵、白芥末和辣根K2P(Kimura 2-parameter)遗传距离在0.088～0.171,均大于0.01,种间变异位点有36个,初步推断利用ITS2序列能将山葵、白芥末和辣根3物种区分开来。另外,GenBank中获得山葵以及近亲西北山嵛菜、辣根、芥末的ITS2序列,MEGA7.0进行种间序列分析,计算K2P,并用邻接法(Neighbor-joining,NJ)构建进化树,山葵与近亲西北山嵛菜聚为一支,棕芥末与白芥末、黑芥末聚为一大支,而辣根单独为一支。通过分析ITS2序列,发现山葵与近亲西北山嵛菜、辣根、芥末的种间K2P遗传距离在0.030～0.105,均大于0.01。研究表明,利用ITS2技术可以鉴别山葵、辣根和芥末等近缘物种,此技术可以用于芥辣类调味品特定原料成分鉴定及定量,为食品质量控制和食品安全提供科学依据。     

Reliability of Listeria monocytogenes Identification by Specific PCR Assessed by Phenotypic and Genotypic Techniques

This study evaluates the possibility of using polymerase chain reaction (PCR) for rapid identification of food-borne Listeria monocytogenes as an alternative to API Listeria system and estimates the incidence of API Listeria misidentifications in food-borne Listeria species. A total of 198 strains, 11 L. monocytogenes, 28 other Listeria species, and 159 food isolates were phenotypically and genotypically characterized by API Listeria profiles and randomly amplified polymorphic DNA (RAPD) profiles, respectively. They were also tested for PCR amplification using genus- and species-specific primers. Clustering analysis of phenotypic and genotypic data showed discrepancies in species identification of some isolates by API Listeria profiles. Their identities were confirmed by 16S rDNA sequencing, and thus, it was revealed that 33% of Listeria innocua and 19% of Listeria welshimeri were misidentified as L. monocytogenes by API Listeria profiles. Reliable identification of L. monocytogenes was obtained by LM1–LM2 specific primers which allowed PCR amplification only in reference strains and isolates previously identified as L. monocytogenes by RAPD and 16S rDNA sequence analysis. These results corroborate the suitability of specific PCR as a rapid and accurate test for the identification of L. monocytogenes, avoiding misidentification with other Listeria species commonly found in food products.     

Molecular identification of dried squid products sold in China using DNA barcoding and SYBR green real time PCR

ABSTRACT

Dried squid products are popular in China as a snack food, side dishes, or refreshments, and the market appeal can be reflected by the high price that occasionally reaches 497 RMB per kg. However, the absence of harmonisation around the definition of squid, as well as the problems with visual inspection for processed seafood products, make alternative species substitution for dried squid products a frequent occurrence. The aim of the present study was to apply a DNA barcoding approach for species identification of 48 dried squid products collected from the largest online shopping platform in China. Moreover, we also developed a novel SYBR green real-time PCR assay (simplex and duplex followed by a melting curve analysis) specific for Illex argentinus and Todarodes pacificus based on cytochrome C oxidase subunit I (COI) gene.

Results highlighted the successful DNA extraction and PCR amplification of a 655 bp COI gene fragment from all products. A maximum similarity value in the range of 98-100% was obtained for all readable sequences using the BOLD and BLAST public databases and four species (Dosidicus gigas, Uroteuthis edulis, I. argentinus, and T. pacificus) were identified. The specificity of the designed primer sets was confirmed against 23 non-target species, and the newly developed methods were successfully applied to screen I. argentinus and T. pacificus in dried squid products.

Overall, DNA barcoding is a robust tool for seafood species identification and the novel method is effective in screening I. argentinus and T. pacificus in food products.     

PCR-RFLP of the mitochondrial cytochrome oxidase gene: a simple method for discrimination between Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

A DNA-based method (PCR-RFLP) has been developed for discrimination between Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). The polymerase chain reaction (PCR) was used for amplification of a 464 bp fragment of the mitochondrial cytochrome oxidase subunit II (COII) gene. Digestion of the products with endonucleases Nci I and Sau 3AI, followed by agarose gel electrophoresis of the digested products, yielded specific restriction profiles that enabled direct visual identification of the species analysed. This PCR-RFLP methodology allowed clear discrimination of Atlantic salmon and rainbow trout samples both in raw and smoked products. © 1999 Society of Chemical Industry     

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     

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis

The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species.The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232 bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.     

Species identification and phylogenetic relationships among five species of psocids from Chinese herbal medicines

The main objectives of the study were to establish an accurate multiplex PCR identification technique for five species of psocids from Chinese herbal medicines and to discuss the phylogenetic relationships among those species. Five species of booklice (Liposcelis bostrychophila, L. entomophila, L. tricolor, L. pearmani and L. rufa) were collected from Chinese herbal medicines. Genomic DNA of individual booklice was obtained via a nondestructive DNA extraction method. The internal transcribed spacer (ITS)1-5.8S-ITS2 gene sequences were obtained by PCR amplifying and sequencing. The primers were designed, and the phylogenetic trees were constructed using the NCBI website and MEGA software. In this research, a Multiplex PCR identification technique was established based on the ITS2 gene for the five species of booklice. The phylogenetic relationships among the five booklice species were analyzed and discussed based on the ITS gene sequences.     

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.     

External Shape Differences between Sympatric Populations of Commercial Clams <Emphasis Type="Italic">Tapes decussatus</Emphasis> and <Emphasis Type="Italic">T. philippinarum</Emphasis>

The actual Italian production of clams is chiefly sustained by the native Tapes decussatus and the fortuitously imported Tapes philippinarum. Both species are commercialized as “Vongola verace”, but the commercial value of T. philippinarum is lower. The discrimination of species by sight is usually difficult and it cannot be done by observation based on shell morphology but only when animals open their valves hence displaying the two siphons. In this study, we propose a new, noninvasive method to discriminate individuals of both species based on the analysis of the external shape of their shells. Accordingly, in sympatric populations at two sites of the Po river outlet, we have chosen individuals (63 for T. decussatus and 57 for T. philippinarum) of comparable commercial size for which a certain genetic discrimination was previously done. Pictures of the left side valve were taken for all specimens. Their profiles were analyzed with the elliptic Fourier analysis. The mean outline for each species was graphically extracted. The coefficients of the harmonic equations were analyzed by multivariate classification (partial least squares discriminant analysis [PLSDA]). Results showed a high percentage of correct classification of individuals of both species (96.6%). Contour analysis reflected the overall shell shape and thus identified morphological aspects that were difficult to recognize and quantify in sight. The high percentage of correct classifications obtained by combining the analysis of elliptic Fourier harmonics with PLSDA demonstrated the feasibility of this method to discriminate species with a high level of resemblance.     

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.