PCR-RFLP Analysis of the Internal Transcribed Spacer (ITS) Region for Identification of 3 Clam Species

ABSTRACT Restriction site analysis of the internal transcribed spacer (ITS) region amplified by the polymerase chain reaction (PCR) was used for the specific identification of 3 clam species: Ruditapes decussatus (grooved carpet shell), Venerupis pullastra (pullet carpet shell), and Ruditapes philippinarum (Japanese carpet shell). PCR amplification using primers based on nucleotide sequences of Mytilus ITS regions produced a fragment of 1195 bp in R. decussatus, 1074 bp in V. pullastra, and 1188 bp inR. philippinarum. Digestion of the PCR products with endonucleases HinfI and Rsa I, followed by agarose gel electrophoresis of the digested products, yielded specific restriction profiles that enabled direct visual identification of the species analyzed.     

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     

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.     

Identification of Nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck fish (Polyprion americanus) by polymerase chain reaction-restriction fragment length polymorphism of a 12S rRNA gene fragment

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the 12S rRNA gene has been used for the specific identification of Nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck fish (Polyprion americanus). Amplification of DNA isolated from muscle samples was carried out using a set of primers flanking a region of 436 bp from the mitochondrial 12S rRNA gene. Digestions of the PCR products with RsaI and Sau96I endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of each species analyzed.     

Microbiological responses to depuration and transport of native and exotic clams at optimal and stressful temperatures

The microbiological responses of two bivalves species from Tagus estuary, Venerupis pullastra (native clam) and Ruditapes philippinarum (exotic clam) were investigated during 48 h of depuration and subsequent simulated transport in semi-dry conditions at two temperatures (4 and 22 °C) until reaching 50% lethal time (LT50). Regardless of temperature and species, the maintenance of clams in water for 48 h (depuration period) did not affect LT50 during transport. R. philippinarum showed higher survival rates than V. pullastra, always reaching LT50 later, especially at 4 °C. Significant differences between clams' species were found in almost all microbiological parameters. This can be related with clams' biological activity and habitat environmental conditions since both clams do not coexist in Tagus estuary. Depuration was efficient to reduce the bacterial load, particularly Escherichia coli, but not efficient to remove Vibrio spp. In both species, the growth of Vibrio spp. was inhibited at 4 °C, whereas exponential growth occurred at 22 °C. Total viable counts significantly increased in most treatments, while E. coli counts significantly decreased to undetected levels, except for non-depurated R. philippinarum simulated transported at 4 °C. Thus, this study highlights the importance of clams depuration for at least 24 h in polluted estuarine areas, followed by transport at low temperatures (4 °C).     

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     

Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene

Adulteration of high quality meat and meat products with their inferior/cheaper counterparts is a problem in the meat industry. The present study investigated the use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial 12S rRNA gene for identification of the origin of meats. PCR-RFLP was applied for species identification of beef, buffalo meat, mutton and chevon. PCR amplification yielded a 456-bp fragment in each of these species. The amplicons were digested with AluI, HhaI, ApoI and BspTI restriction enzymes resulting in a pattern that could identify and differentiate each of the above species. This technique did not yield satisfactory results with meat mixtures/meats. However, consistent results were obtained with both fresh and processed meat samples.     

Sequence analysis of mitochondrial 12S rRNA gene can identify meat species

In this study, sequence analysis of mitochondrial 12S rRNA has been applied for meat species identification. The procedure involves polymerase chain reaction (PCR) amplification of a fragment of mitochondrial (mt) 12S rRNA gene and sequencing of amplicons. Amplified product of mt 12S rRNA gene was 456 bp in size. Species sequenced include cattle (Bos indicus), buffalo (Bubalus bubalis), sheep (Ovis aries), goat (Capra hircus) and mithun (Bos frontalis). Sequences were compared with the reported sequences of low land anoa (Bubalus depressicornis), yak (Bos grunniens) and pig (Sus scrofa). There was no effect of routinely used additives or cooking temperature (72, 90, 120 and 180?°C) on the efficacy of PCR amplification. The closely related species like cattle and buffalo, sheep and goat could also be differentiated decisively by sequence analysis. Sequencing and analysis of mt 12S rRNA gene was, hence, found to be an ideal, authentic and unambiguous qualitative method for meat species identification.     

Differentiation of sturgeon species by PCR-RFLP

A method for identification of sturgeon species in caviar has been developed based on the amplification of a region of the mitochondrial genome (tRNA^Glu/cytochrome b) using the polymerase chain reaction (PCR). To distinguish between several types of sturgeon caviar the obtained 462bp long PCR-products were cut with different restriction endonucleases (RE) resulting in species-specific restriction fragment length polymorphisms (RFLP). The method is suitable to differentiate between 10 species of Acipenser and Huso originating from Europe and Asia.     

Putative virulence genes of Vibrio cholerae from seafoods and the coastal environment of Southwest India

Shrimp, clam and oysters were obtained at two fish markets and at a fish landing dock, and plankton, water and sediment samples were obtained from four river estuaries, in southern India. The samples were analyzed for Vibrio cholerae by conventional isolation techniques and by polymerase chain reaction (PCR) procedures. V. cholerae was isolated from 2 of 5 shrimp, 2 of 5 clam and 5 of 20 water samples. All biochemically confirmed isolates of V. cholerae were positive for toxR. For direct detection of V. cholerae in enrichment broths, PCR was performed using lysates from 0 and 6 h enrichments. All the V. cholerae isolates and enrichment broth lysates were subjected to PCR analysis for the detection of the genes toxR, ctxA, tcpA, ompU, hly, ace, Nag-ST (stn/sto), and ompU. Enrichment broths of all the samples which yielded V. cholerae were positive for toxR, OmpU and hlyA genes, while one of a fresh fish market sample was positive for the ace gene. Choleragenic V. cholerae were absent from all environmental samples and fresh fish from the markets, but one sample of shrimp was positive for V. cholerae O139.     

Temperature gradient gel electrophoresis of the amplified product of a small 16S rRNA gene fragment for the identification of Listeria species isolated from food

The development of a rapid method for the identification of Listeria spp. is described. It is based on the polymerase chain reaction amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis. Forty-five strains of Listeria spp. (Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) were used for the optimization of the protocol. No differences were observed between the results of the identification of the strains tested using traditional methods and those obtained by polymerase chain reaction-temperature gradient gel electrophoresis analysis.     

一对同时鉴定8种动物源性成分的通用引物的制备及应用

肉类真假鉴定是食品检测工作的内容之一,目前已有多种基于PCR的肉类鉴定方法,但是鉴定种类和效率受限。本研究设计了一对基于普通PCR技术可同时鉴定8种动物源性成分的通用引物并建立了鉴定方法。该引物以线粒体DNA为靶标,利用扩增产物中不同物种间的插入缺失多态性片段大小即可鉴定山羊、绵羊、鹿、水牛、牛、牦牛、猪和骆驼8个物种,扩增后分别得到728 bp、704 bp、504 bp、453 bp、448 bp、431 bp、396 bp和326 bp的片段,每种PCR产物经SspI酶切后产生数量和大小不同的片段,可以进一步清晰鉴别8个物种。引物特异性测试表明和其他常见肉类动物DNA无交叉反应,DNA检测最低限度在0.01~0.05 ng。应用本方法对40份市场肉类及产品的检测表明,羊肉串、羊肉卷以及特色畜产品如驼肉、鹿肉和驴肉存在较多的掺假行为。与其他现有PCR检测方法相比,该方法具有简便易行和高通量的优点,可以作为肉类掺假筛选检测的常规方法。     

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.     

A histological study of the transit of Cryptosporidium parvum oocysts through clams (Tapes decussatus)

A histological study was carried out to investigate the transit of Cryptosporidium parvum oocysts through the clam Tapes decussatus. Spat of approximately 5-7 mm shell length were maintained in a tank of natural sea water contaminated with purified C. parvum oocysts. The experiment lasted 240 h and, every 24 h, five specimens were killed, placed in Bouin's fixative, and processed routinely for histological examination. Sections (3 mum) cut from the all body tissues were stained with modified Gomori's trichrome for their accurate identification; the oocysts were detected by a direct immunofluorescence procedure. Oocysts were detected in siphons, gills, stomach, digestive diverticula, and intestine. The oocysts present in the intestine were free or mixed with the intestinal contents; therefore release of these oocysts with the feces should favour dissemination of contamination. Oocysts were found in branchial mucus and within the interfilamentary spaces, which suggests the occurrence of repeated filtrations and the possibility that the retained oocysts maintain their infective capacity.     

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.     

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     

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.     

主要进出口经济鱼类及其制品分子鉴定技术的研究进展

随着我国进出口贸易的不断发展,进出口鱼类品种繁多,数量巨大。但部分国内外企业受高额利润驱使,以假充真、以劣充好事件时有发生,鱼类制品更是真伪难辨,掺假状况极其普遍。通过形态学和生物学方法已经不能满足对鱼类种属鉴定。近年来,随着分子生物技术的迅猛发展,分子检测技术在鱼类品种鉴定领域得到广泛应用。本文针对不同分子鉴定技术进行阐述,包括普通聚合酶链反应(polymerase chain reaction,PCR)技术、多重PCR技术、实时定量PCR技术、DNA条形码技术、环介导等温扩增检测技术(loop-mediated isothermal amplification,LAMP)、聚合酶链反应-限制性酶切多态性技术(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)以及随机扩增多态性DNA检测技术(random amplified polymorphic DNA),并根据不同检测技术的特点进行分析,为进出口鱼类鉴定提供技术支撑。     

Detection of horse DNA in food and feedstuffs using a polymerase chain reaction assay

BACKGROUND: Development of accurate methods for rapid identification of animal materials in food and feedstuffs is essential to protect consumers and also to enforce feed bans. The aim of this study was to develop a polymerase chain reaction (PCR) assay for the specific detection of horse DNA in food and feedstuffs. RESULTS: The primers designed amplified a horse‐specific fragment of 114 bp of the mitochondrial 12S ribosomal RNA gene. The specificity of the primers was verified by PCR analysis of DNA from 32 non‐target species including mammals, birds, fish and plant species. The PCR assay developed allowed the detection of raw and heated horse tissues in muscle/oats mixtures even when the concentration of horse‐derived materials was reduced to 1 g kg^?1. CONCLUSION: The performance of the method was not affected by prolonged heat treatment (up to 133 °C for 20 min at 300 kPa), and consequently it could be very useful in verifying the origin of raw materials in food and feedstuffs submitted to denaturing technologies, for which other methods cannot be applied. Copyright © 2009 Society of Chemical Industry     

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.