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

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

Genetic differentiation between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR–RFLP analysis of a 12S rRNA gene fragment

PCR–RFLP analysis was applied to the identification of two closely related flatfish species: sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Amplification of DNA isolated from fish muscle samples was carried out using a set of primers flanking a region of 321 base pairs (bp) from the mitochondrial 12S rRNA gene. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for AciI and MwoI endonucleases. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with AciI and MwoI enzymes allowed the unequivocal identification of sole and Greenland halibut species. © 2000 Society of Chemical Industry     

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

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

Differentiation of animal species in food by oligonucleotide microarray hybridization

Oligonucleotide microarray hybridization analysis of polymerase chain reaction (PCR) products from the mitochondrial cytochrome b gene DNA was applied to identify different animal species in meat and cheese food samples. A pair of universal primers binding to conserved regions of the vertebrate mitochondrial cytochrome b gene was used to amplify a 377 bp fragment with internal regions of high inter-species variability. PCR products of cattle, pig, chicken, turkey, sheep and goat were unequivocally identified by hybridization with species-specific probe sequences immobilized on an oligonucleotide microarray. In meat samples, 0.1% admixtures of beef or chicken meat were still detectable. By using this new PCR-based DNA chip hybridization for the analysis of 24 commercial food samples from routine control, the simultaneous species composition of mixtures with up to four different species could be determined in a single experiment. The results agreed well with those from the reference methods performed at the local food control authority, which are a combination of enzyme-linked immunosorbent assay (ELISA), species-specific PCR and PCR–RFLP (restriction fragment length polymorphism). Thus, the DNA chip hybridization analysis of cytochrome b PCR products offers a new way for rapid and sensitive species differentiation in food.     

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.     

Development of a method for genetic identification of four species of anchovies: E. encrasicolus, E. anchoita, E. ringens and E. japonicus

Cytochrome b has been successfully employed for genetic identification of four species of anchovies (Engraulis spp) using two methodologies: PCR–RFLP and FINS. The first method allowed the identification of Engraulis anchoita, Engraulis ringens and Engraulis japonicus–Engraulis encrasicolus. In some cases, with a determined restriction profile, this technique was able to differentiate E. japonicus from E. encrasicolus. The second method allowed the identification of those four species and demonstrates that FINS is a suitable technique for the identification of all species studied in this work. Phylogenetic trees show that sequences of E. encrasicolus are grouped into two different clusters. These results are consistent with the previously published data which suggest that some species of genus Engraulis could be cryptic species, being one specie or population distributed in the oceanic habitat and the other one around the coast.     

Distribution of Candida in the oral cavity and its differentiation based on the internally transcribed spacer (ITS) regions of rDNA

This study aimed to determine the distribution of Candida species in the oral cavity and differentiate the species based on PCR amplification, including HinfI and MspI digestion, in order to assess the effectiveness of using the rDNA region for species identification. Samples from saliva as well as palate, tongue and cheek mucosa surfaces were collected from 45 individuals, consisting of three groups: periodontal disease patients; denture‐wearers; and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for colony‐forming units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction, based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI‐5.8S‐ITSII PCR product was digested by HinfI and MspI restriction enzymes. The microbial loads on all sites of the denture‐wearers were found to be significantly higher than control, while in the periodontal disease group only the microbial loads on the tongue were significantly higher than control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to those of seven control species, allowing the differentiation of all seven species and the identification of 14 species from the clinical samples. The MspI restriction digest was not able to distinguish between C. albicans and C. dubliniensis, whereas the HinfI digest could not distinguish between C. tropicalis and C. parapsilosis. It was concluded that PCR–RFLP of the candidal rDNA region has potential for species identification. This study demonstrates the potential use of candidal rDNA as a means for identifying Candida species, based on genotype. The results also indicate the possibility of constructing genetic probes that target specific restriction fragments in the ITSI‐5.8S‐ITSII region, enabling swift and precise identification of Candida species. Copyright © 2012 John Wiley & Sons, Ltd.     

Genetic typification by pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) of wild <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> wine strains

Genetic typification of 120 bacterial isolates of Lactobacillus plantarum and Oenococcus oeni from different Rioja musts and wines was performed by numerical analysis of pulsed-field gel electrophoresis (PFGE) patterns with endonuclease SfiI, and 46 of them were also studied by randomly amplified polymorphic DNA (RAPD)-PCR. A comparative study of both typification methods applied to L. plantarum and O. oeni oenological strains was performed. Bacterial species was determined both by biochemical identification methods and by specific PCR analysis. A wide variety of restriction digest patterns were detected by PFGE among L. plantarum strains (36 unrelated patterns and one closely related pattern, out of 48 isolates), as well as among O. oeni strains (18 unrelated patterns out of 72 isolates). PFGE was shown to be a suitable method for strain differentiation and to determine which strains are present in wine fermentations, with a discriminatory power to type L. plantarum and O. oeni strains higher than that of RAPD-PCR.     

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     

Improved gap‐repair cloning method that uses oligonucleotides to target cognate sequences

In vivo or gap‐repair cloning in yeast has been widely recognized as one of the most efficient means for error‐free construction of plasmids. A protocol is described here that allows easy and efficient gap‐repair cloning that is based on two major modifications. Instead of subcloning, the targeting plasmids are constructed using oligonucleotides from sequences derived from the upstream and downstream sequences of the fragment to be cloned. These sequences are selected so that they can lead to the generation of recognition sites for restriction enzymes that produce blunt ends. Accordingly, this procedure can be applied to any DNA fragment, regardless of whether these include unique restriction sites to generate the targeting ends. With the strategy described, ～50 bp upstream and downstream targeting ends are generated that allow efficient cloning. Further, to allow easy identification of the positive clones, the annealed oligonucleotides are cloned in frame with the lacZ fragment present in the plasmid. Accordingly, these plasmids produce blue Escherichia coli colonies on media containing X‐Gal. On the other hand, plasmids rescued from yeast that have acquired the respective cognate sequences produce white colonies. To demonstrate the efficiency of the method, this report includes the cloning of fragments harbouring the CDC28, CAK1, CIN5 and CLB2 genes. We found that 30–100% of the analysed plasmids carried the expected inserts. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Differentiation of fish species by PCR-based DNA analysis of nuclear genes

Food control laboratories are confronted with new challenges in respect to fishery product authentication; examples are identification of hybrids (e.g. in case of catfish, tilapia, sturgeon, snapper) and assignment of fish to certified stocks. Against this background, differentiation of fish species and populations by polymerase chain reaction (PCR)–based DNA analysis of nuclear genes has become of considerable importance as a tool for the completion of mitochondrial gene analysis. Four applications of nuclear gene analysis are presented: (1) fish species identification using the intronless rhodopsin RH1 gene; (2) detection of “fish” as an allergenic foodstuff by means of universal primers amplifying a segment of a parvalbumin gene; (3) differentiation of cod (Gadus morhua) from various fishing grounds by exon-primed intron-crossing PCR of a parvalbumin gene intron; (4) detection of hybridization between North Atlantic redfish species (genus: Sebastes) by restriction fragment length polymorphism analysis of amplicons obtained from the second intron of the 7S ribosomal protein gene.     

Molecular identification of cephalopod species by FINS and PCR-RFLP of a cytochrome b gene fragment

Identification of commercial species is a relevant issue to assure the correct labeling of seafood products. In this work two different molecular techniques, FINS (Forensically Informative Nucleotide Sequencing) and PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) were developed to identify 8 cephalopod species (families Loliginidae and Ommastrephidae) employing a fragment of the cytochrome b gene. DNA amplification for all of the species was carried out with a new set of specific primers designed in this study for cephalopods. FINS is a technique based on DNA sequencing, while PCR-RFLP allows direct species identification by comparing specific DNA restriction patterns. Both techniques are useful for cephalopod species identification. 17 food products (mainly "squid rings") were analyzed and the species employed for their manufacture identified by FINS.     

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

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

A rapid and high-throughput real-time PCR assay for species identification: application to stockfish sold in Italy

In some parts of Italy, there is a tradition of eating special, highly prized species of cod, fished and dried in Norway. In order to safeguard the value of this food product, in 2005, the Italian government legislated that the commercial term “stockfish” can only be attributed to Gadus morhua (Gm). In this study, we present an improved real-time PCR assay for efficient identification of Gm with respect to other gadiforms of commercial interest. The method is applied to 437 stockfish samples, collected in various Italian regions, in order to verify whether labelling regulations had been respected and to report instances of fraud in the Italian stockfish market. The PCR method employed here allowed rapid and economical identification of the species, with a very high percentage of correct identifications.     

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。     

Persistence of Listeria monocytogenes strains isolated from products in a Polish fish-processing plant over a 1-year period

Seventy-one presumptive Listeria monocytogenes strains were isolated over a year from 152 samples comprising raw fish (salmon, seatrout) and their products (mainly, vacuum-packed cold-smoked sliced salmon) in a selected Polish fish-processing plant. Contamination of raw materials was at the level of 4.3–15.4%, whereas final products revealed significantly higher contamination (up to 77.8%) than regarded by other studies as typical (up to 40%). Strains were identified using conventional microbiological methods (including API®LISTERIA tests) and the PCR technique (aimed at iap gene fragment detection). A random amplification polymorphic DNA (RAPD) technique was applied to analyse their intraspecies diversity. RAPD typing revealed an incidence of eight RAPD types. Three of them were isolated over 8–10 months during the plant monitoring. It suggested that they were a persistent element of ‘in-house’ microflora and the applied typing technique produced evidence that fish products could be probably contaminated at the last stages of fish processing (e.g. smoking, slicing, and/or packaging). Their occurrence was probably supported by clone selection caused by ineffective application of cleaning and sanitizing procedures. The possibility of colonization of the production environment by fish-originated L. monocytogenes was also proven. Strains that belonged to a dominant RAPD type were additionally subjected to restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE). RFLP-PFGE confirmed intraspecies similarity of strains belonging to a dominant RAPD type. A subset of strains from salmon samples was also characterized by serotyping. Contrary to earlier reports, they belonged mainly (91.7%) to the serotype 4.     

DNA Arrays and Membrane Hybridization Methods for Screening of Six Lactobacillus Species Common in Food Products

Dot blot and deoxyribonucleic acid (DNA) array hybridization assays for the traceability of Lactobacillus species in food have been developed to monitor and validate typical food products. A primer set was designed to amplify the 540-bp region located at +157 of the tuf (Elongation factor Tu) gene of the Lactobacillus genus. An oligonucleotide array, containing 73 Lactobacillus species-specific tuf sequences representing 21 species, was developed and tested for identifying L. paracasei, L. rhamnosus, L. plantarum, and L. buchneri. We also tested a rapid screening method for monitoring the species present in airy samples. Dot blot hybridization identified polymerase chain reaction amplicons immobilized on nylon membranes, using six tuf-based cyanine-3-labeled 18-mer oligonucleotides, specific for L. paracasei, L. zeae, L. fermentum, L. plantarum, L. rhamnosus, and L. buchneri. This method discriminates between multiple species of Lactobacilli isolated directly from cheese samples, simultaneously. The tuf gene sequences, verified here with the DNA array method and used in dot blot hybridization, were shown to be a reliable tool for the simultaneous detection and differentiation of four Lactobacillus species. The hybridization techniques developed in this study may be useful in food processing and the analysis of food origin traceability.     

Identification and molecular characterisation of lactobacilli isolated from traditional Koopeh cheese

This study was undertaken in order to phenotype and genotype wild‐type lactic acid bacteria isolated from Koopeh cheese of West Azerbaijan. Lactic acid bacteria (LAB) isolates were identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) and confirmed by phylogenetic analysis. Genotyping based on phylogenetic analysis of 16s rDNA gene revealed that LAB isolated from Koopeh cheese were mostly Lactobacillus plantarum as well as other species including Lactobacillus brevis, Entrococcus faecium and Enterococcus durans. It was concluded that a combined phenotypic and genotypic method could be used as a reliable technique for the identification and differentiation of LAB from dairy products.     

Comparison and evaluation of molecular methods used for identification and discrimination of lactic acid bacteria

BACKGROUND: Lactobacillus and Bifidobacterium strains are present in a great variety of habitats, including fermented products, probiotic concoctions and the human colon. Some species are so closely related that it is difficult to distinguish them by microbiological techniques. Nevertheless, discrimination of isolates is an important issue in respect of application, and molecular methods such as restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) or species‐specific polymerase chain reaction (PCR) might help in resolving this problem. In this study, PCR, RFLP and sequencing were applied to identify lactobacilli and bifidobacteria originating from various sources and the DSMZ strain collection. RESULTS: The microbiological composition of foods was analysed by molecular methods. Using species‐specific PCR primers, three restriction enzymes (AluI, HhaI and RsaI) and sequencing, three Bifidobacterium and six Lactobacillus reference strains could be distinguished and four additional lactobacilli of food origin were identified. CONCLUSION: A combination of three molecular methods resulted in successful discrimination of nine reference strains and four isolates of food origin. Since these methods are not always accurate owing to their high genetic homogeneity, it is advisable to use more than one method for the identification of L. casei and closely related species. Copyright © 2012 Society of Chemical Industry