Polymorphisms in Phytophthora infestans: Four Mitochondrial Haplotypes Are Detected after PCR Amplification of DNA from Pure Cultures or from Host Lesions

Four pairs of primers were designed for PCR amplification of known polymorphic regions of the mitochondrial genome of Phytophthora infestans. Digestion of the amplified products with restriction enzymes allows identification of previously identified haplotypes. Product P2 cut with MspI uniquely identifies haplotypes Ib and IIa, while types Ia and IIb are differentiated by digestion of product P4 with EcoRI. Digestion of products P1 and P3 gave results similar to that with digestion of P4, but amplification of these products was less robust. Thus, all four common haplotypes are identified by amplifying and digesting products P2 and P4. Identification of haplotypes was also possible from DNA extracted directly from small, late-blight lesions on both tomato and potato leaves, making isolation of the fungus unnecessary. A rapid and efficient method of monitoring changes in the pathogen population is facilitated. These PCR primers were also useful for differentiating other Phytophthora species.     

Comparison of Streptococcus thermophilus strains by pulse field gel electrophoresis of genomic DNA

Pulse field gel electrophoresis (PFGE) was utilised to compare the genomes of 16 Streptococcus thermophilus cultures from yoghurt, cheese, laban and dahi after digestion with the restriction endonucleases, SfiI, SmaI and BssHII. PFGE profiles could be used for strain identification and were also useful in predicting relatedness of certain strains. Genetic variations between specific morphotypes of a highly proteolytic culture were not detectable by PFGE in this study. Statistical analysis of SmaI restriction patterns enabled the clustering of strains into two groups which corresponded with biochemical properties of the strains examined and suggested that PFGE profiles could be useful in predicting biochemical characteristics.     

Rapid Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">acidophilus</Emphasis> by Restriction Analysis of the 16S–23S rRNA Intergenic Spacer Region and Flanking 23S rRNA Gene

A rapid molecular approach was developed for the initial identification of Lactobacillus acidophilus strains which are difficult to identify using a single biochemical test. The 16S–23S rRNA intergenic spacer regions and flanking 23S rRNA genes of 19 strains of lactobacilli were amplified and the nucleotide sequences and restriction site polymorphisms were analyzed. AluI was the most useful of the restriction enzymes analyzed and produced reproducible digestion profiles in the L. helveticus, L. plantarum, and L. casei groups, as well as in L. acidophilus. This restriction fragment length polymorphism method may be useful for the identification of L. acidophilus strains in dairy products.     

NotI passporting to identify species composition of complex microbial systems

We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.     

Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens

For restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, the rDNA fragments of 1.5 kb were amplified by polymerase chain reaction (PCR) from crude cell lysates of various methanogenic species which were prepared by a combined technique of ultrasonic treatment and protease digestion. The PCR products were purified by the polyethylene glycol precipitation method and treated with various restriction enzymes. The 16S rDNA fragments digested with HaeIII or HhaI gave species-specific RFLP profiles on simplified agarose gel electrophoresis. 16S rDNA gragments of 0.4 kb from the bulk DNA extracted from mixed populations of anaerobic sludge were also amplified by PCR with a pair of methanogen-specific primers and cloned directly by the T-A cloning technique. The cloned 16S rDNAs from recombinants were reamplified by PCR, and RFLP pattern analysis was performed following digestion with HhaI. The PCR-RFLP analysis of 16S rDNA with the present protocol can be completed within one day, provided that sufficient amounts of test cells are available, and has great promise as a simple and rapid technique for identification of methanogens. A combined method consisting of PCR amplification, direc cloning with T vectors, and RFLP analysis of 16S rDNA is also useful for rapid estimation of the mixed population structure of methanogens without the need for cultivation and isolation.     

Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris

The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis. Amplicons of 265 bp and 447 bp of htrA and yueF, respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with TaqI produced DNA bands of 90 bp and 175 bp with ssp. lactis, and 66 bp and 199 bp with ssp. cremoris. Similarly, restriction of PCR product of 447 bp size with AluI produced digested fragments of 125 bp and 322 bp with ssp. lactis, and 71 bp and 376 bp with ssp. cremoris. The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with TaqI and AluI, respectively.     

Stable MSAP Markers for the Distinction of Vitis vinifera cv Pinot Noir Clones

Grapevine is one of the most economically important fruit crops. Molecular markers have been used to study grapevine diversity. For instance, simple sequence repeats are a powerful tool for identification of grapevine cultivars, while amplified fragment length polymorphisms have shown their usefulness in intra-varietal diversity studies. Other techniques such as sequence-specific amplified polymorphism are based on the presence of mobile elements in the genome, but their detection lies upon their activity. Relevant attention has been drawn toward epigenetic sources of variation. In this study, a set of Vitis vinifera cv Pinot noir clones were analyzed using the methylation-sensitive amplified polymorphism technique with isoschizomers MspI and HpaII. Nine out of fourteen selective primer combinations were informative and generated two types of polymorphic fragments which were categorized as “stable” and “unstable.” In total, 23 stable fragments were detected and they discriminated 92.5 % of the studied clones. Detected stable polymorphisms were either common to several clones, restricted to a few clones or unique to a single clone. The identification of these stable epigenetic markers will be useful in clonal diversity studies. We highlight the relevance of stable epigenetic variation in V. vinifera clones and analyze at which level these markers could be applicable for the development of forthright techniques for clonal distinction.     

Genome analysis ofPropionibacterium freudenreichii by pulsed-field gel electrophoresis

Preparations of intact genomic DNA from 23 strains ofPropionibacterium freudenreichii were compared by digestion with restriction endonucleases and subsequent transverse alternating field electrophoresis (TAFE). Seven restriction enzymes,AsnI,DraI,HpaI,SnaBI,SpeI,SspI, andXbaI, produced DNA fragments useful for strain comparisons. A characteristic restriction fragment pattern was identified for 18 of the 23 strains. Estimates for the genome size of theP. freudenreichii strains ranged from 1.6×10⁶ to 2.3×10⁶ base pairs based on the sum of fragment sizes obtained with restriction digests. Restriction endonuclease patterns resolved by TAFE are useful for strain identification.     

Sequence analysis of the gene encoding H antigen in <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from food in Morocco

In order to develop other molecular method useful for typing of motile and non motile Escherichia coli strains, a total of 207 strains of E. coli (133 reference strains, 74 food strains) were characterized by analysis of sequences of their amplified flagellin-encoding (fliC) gene products. The collection of reference strains was used for database building of fliC gene sequences. Application of this identification system to 74 E. coli food isolates revealed a reproducible and clear cut classification with very good correlation to results obtained by HhaI restriction of the amplified flagellin gene. The proposed determination of fliC sequences variations should be helpful for epidemiological studies.     

Molecular authentication of <Emphasis Type="Italic">Tetrastigma hemsleyanum</Emphasis> from its adulterant species using ISSR,CAPS, and ITS<Subscript>2</Subscript> barcode

Tetrastigma hemsleyanum is a rare and endangered herb, which is commercialized as the resource of anti-cancer drugs. Wild T. hemsleyanum plants are on the verge of extinction recently, there are increasing numbers of counterfeits on the market. In the present study, inter-simple sequence repeat (ISSR), Cleaved amplified polymorphic sequence (CAPS), and the internal transcribed spacer region II (ITS₂) barcode were used for the first time for the authentication of T. hemsleyanum from its commonly counterfeits. ISSR analysis suggested that it was a useful method for distinguishing T. hemsleyanum from its adulterants of different genus. However, it was insufficient to distinguish T. hemsleyanum from those adulterants of the same genus. ITS₂ of T. hemsleyanum and the commonly counterfeits were amplified and sequenced. The Neighbor-Joining tree constructed from the ITS₂ sequences showed that T. hemsleyanum was clearly differentiated from all counterfeits samples. A mutation site in the ITS₂ region of T. hemsleyanum had been found which could be recognized by the restriction endonuclease NcoI. T. hemsleyanum could be readily distinguished from counterfeits as the PCR products from T. hemsleyanum could be digested sufficiently by NcoI, while the PCR products from counterfeits could not be digested. The results indicated that CAPS and ITS₂ barcode methods provided effective and accurate identification of T. hemsleyanum from all its adulterants, while ISSR could only distinguish T. hemsleyanum from its adulterants of different genus. The CAPS method developed in the present study will serve as a reliable tool for safe and effective use of T. hemsleyanum in the clinic application. It will also play an important role for the identification, management and conservation of this endangered species.     

Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

RFLP-PCR analysis of the aroA gene as a taxonomic tool for the genus Aeromonas

The aroA gene has been identified as a target in screening for the presence of most Aeromonas species so far described by PCR. Synthetic oligonucleotide primers of 24 and 25 nucleotides were used by PCR assay to amplify a sequence of the aroA gene, which encodes 3-phosphoshikimate-1-carboxyvinyltransferase, a key enzyme of aromatic amino acids and folate biosynthetic pathway. A 1236-bp DNA fragment, representing most of the aroA gene, according to the nucleotide sequence of A. salmonicida, was amplified from all Aeromonas species tested, which represented most of the 14 hybridization groups. HaeII digestion of the 1236-bp fragment generated a restriction fragment length polymorphisms which could be used as a powerful tool for identification of aeromonads to the genus level.     

RFLP of 16S-ITSrDNA Region to Differentiate Lactobacilli at Species Level

The 16S-ITS (internal transcribed spacer) region of the rrn operon was amplified by polymerase chain reaction (PCR). The amplification products were analysed by restriction fragment length polymorphism (RFLP) using a set of restriction enzymes, AluI, HaeIII, and TaqI. Restriction pattern analyses revealed that TaqI restriction enzyme could clearly differentiate the nine reference strains of Lactobacillus used in the study. This revised version was published online in July 2006 with corrections to the Cover Date.     

Accurate identification of three cryptic species of rodents of the genus Calomys using RAPD-PCR and mitDNA RFLP markers

Calomys musculinus, Calomys laucha and Calomys venustus are cryptic species with overlapping distribution ranges. C. musculinus is the natural reservoir of Junin virus (Arenaviridae), the etiological agent of Argentine Hemorrhagic Fever. In epidemiological studies it is very important to unequivocally identify the species of individuals collected in the field in order to test virus infection. The purpose of this work was to describe molecular markers allowing a prompt and clear characterization of individuals of the three species. We studied the D-loop region of mitochondrial DNA by restriction fragment length polymorphism (RFLP). This region was amplified by PCR and the product was digested with nine restriction endonucleases (RE). Eight recognize 4 bp restriction sites (Taqα I, Tsp509 I, Aci I, Rsa I, Alu I, Nla III, Hae III and Mse I) and one recognized a 6 bp sequence (Ase I). Two of them (Aci I and Hae III) did not distinguish any of the three species. Alu I did not discriminate between C. musculinus and C. laucha, but clearly distinguished both from C. venustus. Taq I did not distinguish C. laucha from C. venustus, but differentiated both species from C. musculinus. Mse I distinguished the three species, but some of the polymorphisms of C. musculinus are very similar to C. laucha's restriction pattern. The enzyme Nla III distinguished the three species, but it is highly polymorphic within species. The enzymes Tsp509 I, Rsa I and Ase I clearly discriminated the three species, and patterns obtained with the three of them are recommended for reliable identification of individuals collected in the field. The same DNA samples were used to obtain Random Amplified Polymorphic DNAs (RAPDs) patterns. Several bands produced with primers A02, A06, A08, A09, B09 and OPA02 are species specific and could also be used for identification.     

Intraspecies Genomic Groups in Enterococcus faecium and Their Correlation with Origin and Pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

Rapid PCR-Based Method Which Can Determine Both Phenotype and Genotype of Lactococcus lactis Subspecies

A highly efficient, rapid, and reliable PCR-based method for distinguishing Lactococcus lactis subspecies (L. lactis subsp. lactis and L. lactis subsp. cremoris) is described. Primers complementary to positions in the glutamate decarboxylase gene have been constructed. PCR analysis with extracted DNA or with cells of different L. lactis strains resulted in specific fragments. The length polymorphism of the PCR fragments allowed a clear distinction of the L. lactis subspecies. The amplified fragment length polymorphism with the primers and the restriction fragment length polymorphism of the amplified products agreed perfectly with the identification based on genotypic and phenotypic analyses, respectively. Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical L. lactis subsp. cremoris fragments.     

Discrimination between invasive Ponto‐Caspian gobies using a PCR‐RFLP method

Accurate identification of invaders, and especially their juveniles and eggs, is a difficult task if several morphologically similar species co‐occur. The aim of the study was to develop and test a rapid and cost‐effective procedure for identification of five species of invasive gobies occurring in the middle Danube basin, namely round goby Neogobius melanostomus, bighead goby Ponticola kessleri, monkey goby Neogobius fluviatilis, racer goby Babka gymnotrachelus and tubenose goby Proterorhinus semilunaris. First, a 708 bp fragment of the cytochrome oxidase I gene was amplified and sequenced for representative samples of these five species. Appropriate sequences of the five species available in public databases were used for in silico analysis. A digestion of the amplified fragment with the BfaI enzyme was found to be suitable for the species identification, as it showed unique restriction patterns for each species. The technique was also successfully applied for fish remains from burbot Lota lota stomachs. Thus the technique could be a useful tool in monitoring biological invasions, especially by identifying specimens that could not be determined on the basis of morphological features. The results demonstrate that the PCR‐RFLP method may in some cases be more reliable for species identification than a standard DNA sequencing.     

PCR amplification and characterization of the intergenic spacer region of the ribosomal DNA in Pyrenophora graminea

Successful amplification of the whole intergenic spacer region of the nuclear ribosomal repeat (IGS) in Pyrenophora graminea was obtained with a PCR-based assay. Single amplification products showed length differences. Depending on the length of the IGS-PCR product, ca. 3.8 or 4.4 kb, two groups of isolates could be identified. The RFLP patterns of isolates obtained with the 6-base cutting enzymes ApaI, BglII, DraI, EcoRV, HindIII and SacI were similar within each group and different between the two groups. Restriction patterns of IGS-PCR products digested with the 4-base cutting enzyme AluI were polymorphic among isolates in spite of their IGS-PCR product length. In order to characterize the long and short IGS-PCR products the restriction map is shown. The long product shows an additional HindIII site and a BglII site that is lacking in the short product. However, the latter shows a SacI site that is not present in the long IGS-PCR product. Therefore, the described PCR-RFLP analysis of the IGS appears to be a useful tool to resolve genetic variation between P. graminea isolates.     

Morel Phylogeny and Diagnostics Based on Restriction Fragment Length Polymorphism Analysis of ITS Region of 5.8S Ribosomal RNA Gene

Thirty-six cultures representing eight Morchella and related genera, namely, Morchella esculenta, M. crassipes, M. spongiola, M. vulgaris, M. angusticeps, M. conica, Mitrophora semilibera and Verpa conica were subjected to restriction analysis of ITS1-5.8SITS2 region of rDNA. Six restriction endonuclease enzymes viz TaqI, EcoRl, Mspl, Rsal, Hinfl and BsuRl were used to generate restriction fragments and analysis of phylogenetic relationships among morels. The Amplified Ribosomal DNA Restriction Analysis (ARDRA) not only distinguished yellow morels from black morels but also separated related genera Mitrophora semilibera and Verpa conica from true morels. Simultaneously, each morel species could be separated from each other exhibiting considerable phylogenetic distances. The unique restriction fragment profiles generated by the restriction endonucleases enabled us to identify marker fragments to distinguish each species within and amongst the morel group. Since no intra-specific variation in restriction profiles by the six restriction endonucleases could be visualized among monospores, the technique could be used for rapid identification of wild morel specimens as a cheap alternative to direct sequencing for germplasm cataloguing.     

Molecular identification of house dust mites and storage mites

Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR–RFLP of ITS2 region. With the established PCR–RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR–RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.