Differentiation of species of the genus Saccharomyces using biomolecular fingerprinting methods

The genus Saccharomyces comprises very closely related species. This high degree of relationship makes a simple identification and differentiation of strains difficult since these species are hardly discriminable by their morphological and physiological features. A sequence analysis of ribosomal DNA and the corresponding internal transcribed spacers can only rarely be successfully applied. In this study, we proved the applicability of a novel DNA fingerprinting method, the SAPD-PCR (specifically amplified polymorphic DNA) and of MALDI-TOF-MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) fingerprinting with the MALDI Biotyper for the differentiation of species belonging to the genus Saccharomyces. It was possible with SAPD-PCR to create specific banding patterns for all Saccharomyces species. Different strains of the same species produced nearly the same banding patterns. Specific and reproducible reference spectra could be generated for each of the strains with the MALDI Biotyper. Therefore, SAPD-PCR and MALDI-TOF-MS can be fast and reliable tools to identify these related Saccharomyces species which are applied in many biotechnological processes.     

Identification of thermophilic and marine bacilli from shallow thermal vents by restriction analysis of their amplified 16S rDNA

AIMS: In order to identify 73 thermophilic isolates from shallow, marine thermal vents of Eolian Islands, we compared their restriction patterns of amplified 16S rDNA with those of nine well described Bacillus species and eight Eolian Bacillus strains. METHODS AND RESULTS: This study allowed to assign 57 (78%) isolates to different Bacillus species. Nineteen field strains were recognised as representatives of four described species, namely B. thermodenitrificans, "B. caldolyticus", B. vulcani and B. stearothermophilus. The profiles of 38 isolates matched instead, those of seven Eolian strains (B. thermodenitrificans strain A2, B. licheniformis strain B3-15, and five novel species, represented by Bacillus strain 1bw, Bacillus strain 4-1, Bacillus strain 5-2, Bacillus strain 10-1, Bacillus strain 1as). Among the 16 unidentified isolates, seven restriction patterns were recognised. CONCLUSIONS: This study showed that restriction analysis of amplified 16S rDNA is useful for a rapid and reliable identification of strains belonging to described species as well as for recognition of new species. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed a high taxonomic diversity among the thermophilic bacilli isolated from Eolian Islands and a distinct distribution of the species within the Eolian hydrothermal vent system.     

Typing of Staphylococcus epidermidis strains by random amplification of polymorphic DNA

Abstract The polymerase chain reaction was used to obtain randomly amplified polymorphic DNA profiles for typing of Staphylococcus epidermidis strains. Epidemiologically unrelated S. epidermidis isolates were screened with randomly amplified polymorphic DNA analysis. The discriminating ability of 45 randomly designed 10-mer primers was assessed. The highest discriminatory power was obtained with the 10-mer oligonucleotide OPAM-12. In typing a total of 13 unrelated S. epidermidis strains with OPAM-12,11 different banding profiles were obtained reproducibly by agarose gel electrophoresis. The discriminatory power of the method with OPAM-12 was estimated using the D value of Hunter and Gaston (1988) to be 0.961. A reproducibility index of 1 was obtained after typing a total of 40 cultures including 12 triplicates and one quadruplicate of the 13 unrelated strains. Following the described procedure, the randomly amplified polymorphic DNA method provided a rapid, simple and reproducible alternative to other S. epidermidis typing systems.     

Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.     

Differentiation of <Emphasis Type="Italic">Vaccinium</Emphasis> Cultivars and Wild Clones Using RAPD Markers

The potential use of the random amplified polymorphic DNA (RAPD) technique for characterization and assessment ofgenetic relatedness was investigated in 13 wild cranberry (Vaccinium macrocarpon Ait) clones collected from Newfoundlandand Labrador, Canada. RAPD markers were also used to distinguish representatives of three different Vaccinium species.Twenty-two decamer arbitrary primers yielded informative, reproducible and polymorphic banding patterns in 13 cranberryclones and produced a total of 134 bands, of which 114 were polymorphic. The UPGMA cluster analysis separated the clonesinto two main groups: Cluster I of seven and Cluster II of six with 0.62 to 0.91 and 0.50 to 0.77 Nei and Li’s similarity range,respectively. In another experiment, a subset of eight primers was used and the RAPD markers discriminated the threeVaccinium species: cranberry, lowbush blueberry (V. angustifolium Ait) and lingonberry (V. vitis-idaea L). The RAPDpolymorphisms will be useful for Vaccinium genotype differentiation and the technology should be valuable for themaintenance of germplasm banks and the efficient choice of parents in the current Vaccinium germplasm improvementprogram.     

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.     

RAPD typing for distinguishing species and strains in the genus Listeria

The randomly amplified polymorphic DNA (RAPD) technique was employed in the development of a typing protocol for Listeria isolates, particularly Listeria monocytogenes strains. A single strain of L. monocytogenes was used and 200 random decamer primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. Three candidate primers displaying potentially useful banding patterns were selected and tested against 52 L. monocytogenes strains, encompassing 11 serotypes, and 12 other strains representing five other Listeria spp. Thirty-four banding profiles were obtained with one particular primer. RAPD analysis allowed differentiation between Listeria spp. and was found to further subdivide strains of the same serotype. Where only one primer was used strains from different serotypes were occasionally found to produce identical banding profiles. RAPD analysis, which in our hands proved to be reproducible, shows much promise as a molecular alternative to traditional L. monocytogenes typing protocols.     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

应用RAPD技术鉴定地霉的实验研究

目的探讨RAPD技术在快速鉴定地霉中应用。方法用E.Z.N.A.yeastDNAkit提取地霉菌基因组DNA,采用随机引物AP3(5'-TCGTAGCCAA-3')、ATG(5'-ATGGATCGGC-3')、RP2(5'-AAGGATCAGA-3')、OPA-10(5'-GTGATCGCAG-3')对临床上致病性白地霉、林生地霉皮损株和血液株的基因组DNA进行扩增,对各病原菌的DNA指纹的特征进行分析。结果成功提取了地霉的基因组DNA,其纯度和浓度均能满足PCR反应的要求。利用4种引物对基因组DNA进行扩增,不同种真菌的DNA显示不同的DNA带型,分离自不同感染部位的同种不同株真菌的DNA显示的主要DNA带型基本一致。结论采用E.Z.N.A.yeastDNAkit提取的地霉基因组DNA可以用于PCR反应。RAPD法鉴定地霉菌简单、快速、特异,可用于临床诊断。     

Detection and Identification of Bursaphelenchus Species with DNA Fingerprinting and Polymerase Chain Reaction

We have evaluated the potential of DNA-based methods to identify and differentiate Bursaphelenchus spp. and isolates. The isolation of a DNA probe, designated X14, and development of a DNA fingerprinting method for the identification and differentiation of Bursaphelenchus species and strains is described. Polymerase chain reaction (PCR) amplification of DNA isolated from Bursaphelenchus species using two primers derived from the sequence of the cloned repetitive DNA fragment X14 resulted in multiple band profiles. A 4-kb fragment thus amplified from B. xylophilus DNA was not amplified from B. mucronatus or B. fraudulentus DNA. In addition to this fragment, several other fragments are amplified from the three species. The banding patterns obtained allowed species identification and may have value in determining taxonomic affinities.     

随机扩增多态性DNA技术在鲍氏层孔菌菌株鉴别中的应用

用20个随机引物对7个不同来源的鲍氏层孔菌菌株进行了RAPD分析．结果表明120个随机引物中,有17个引物的扩增产物DNA条带表现出明显的多态性,不同引物对供试菌株扩增出现的DNA条带数目少则10条,多达33条．DNA片段从250bp到2000bp;采用17个引物对7个鲍氏层孔菌菌株共扩增出DNA片段带377条,不同引物扩增出的DNA片段谱带存在较大差异．采用UPGMA系统聚类法,将7个菌株聚类为两大类,能直观准确地揭示菌株间的差异并加以鉴别．     

The differentiation of Carnobacterium divergens using the random amplification of polymorphic DNA polymerase chain reaction technique

The potential of the randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique to differentiate Carnobacterium divergens from other members of the genus Carnobacterium was examined. A numerical analysis of the genomic profiles obtained demonstrated that it was possible to differentiate the C. divergens strains from other Carnobacterium strains using this technique. The heterogeneity observed in the representatives of the species C. piscicola adds further weight to the suggestion in other taxonomic studies that subspecies of this species exist.     

Evaluation of different DNA-based fingerprinting methods for typing Photobacterium damselae ssp. piscicida

This study evaluates the effectiveness of three different molecular techniques, repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and the random amplified polymorphic DNA (RAPD-PCR) for rapid typing of Photobacterium damselae ssp. piscicida strains isolated from different species of marine fish and geographic areas. The results obtained by the three methods showed that RAPD and ERIC-PCR were more discriminative for suitable rapid typing of Ph. damselae ssp. piscicida than REP-PCR. The analysis of DNA banding patterns generated by both molecular methods (RAPD and ERIC-PCR) clearly separated the strains into two main groups that strongly correlated with their geographic origin. Moreover, the REP-PCR analysis was less reproducible than the RAPD and ERIC-PCR methods and does not allow the establishment of genetic groups. RAPD and ERIC-PCR constitute valuable tools for molecular typing of Ph. damselae ssp. piscicida strains, which can be used in epidemiological studies of photobacteriosis infections.     

Application of ITS sequence analysis, RAPD and AFLP fingerprinting in characterising the yeast genus Fellomyces

Three molecular techniques, ITS sequence analysis, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to study phylogenetic and genotypic relationships among strains of the genus Fellomyces. In the analyses were included strains isolated predominantly from epiphytic lichens collected in Indonesia, China and Mexico. The polyphasic approach indicated that the Fellomyces isolates are genotypically heterogeneous and that lichens represent a specific environment for selection of large number of the sterigmatoconidia producing species. The phylogenetic and genotypic analysis confirmed the existence of 11 currently accepted Fellomyces species and indicated that several species may be the new representatives of the genus. The RAPD and AFLP analyses demonstrated a higher potential in distinguishing the Fellomyces strains than the ITS regions. Since the sequence analysis showed low or no divergence among several strains, both RAPD and AFLP fingerprinting indicated that the strains may be discriminated at the species level.     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch, based on detection of RAPD molecular markers

The gynogenetic silver crucian carp, Carassius auratus gibelio, is a unique model system for studying evolutionary genetics and selective breeding, owing to its specific genetic background and reproductive modes. Five gynogenetic clones were analyzed by the random amplified polymorphic DNA (RAPD) technique, using 30 10-nucleotide-long primers. Twenty-six primers produced well-amplified DNA fragments with reproducible banding patterns, and 24 primers were polymorphic. Nearly identical banding patterns were observed among individuals within each clone, suggesting that each clone might possess a specific pattern owing to its gynogenesis. In contrast, the RAPD patterns of the five clones differed from each other. A phylogenetic tree was constructed using UPGMA cluster analysis based on a total of 3,744 distinguishable fragments (156 per individual). Average genetic distances within and among the five clones clearly indicated their intraclonal homogeneity, interclonal heterogeneity, and phylogenetic relationships. Clones A and P were the most closely related, whereas the most divergence was seen between clone D and clone E or F. A total of 88 polymorphic fragments were scored from 24 primers after excluding bands that were monomorphic for the five clones. Most primers corresponding to the polymorphic fragments amplified reproducible markers specific for one clone or that were shared by two, three, or four clones. Several primers (e.g., Opj-1, Opj-7, and Opp-10) produced abundant banding patterns that could be used to discriminate between the five clones. Markers specific for one or two clones were also identified. The RAPD markers identified in this study will likely benefit evolutionary genetics and selective breeding studies.     

Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis

Several molecular methods are currently available for identification and discrimination of bacterial strains within the same species, which vary in efficiency and required labour. Here we applied a novel method for fingerprinting genomes, called arbitrarily primed PCR (AP-PCR), to the delineation of strains within the species Porphyromonas gingivalis. Using a single primer on a set of nine strains, nine simple distinct banding patterns, indicative of genetic polymorphism, were observed. Common amplicons and amplicons shared by only some strains were also observed, the latter suggesting that AP-PCR can be used to generate polymorphic markers. Genomic fingerprinting obtained by AP-PCR was independent of the quality of DNA. Assays performed directly using whole cells as a source of DNA template indicated that AP-PCR from colony is a quick, simple and accurate procedure.     

GENETIC VARIATION IN ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE): IS IT LINKED TO FREQUENT EPIDEMICS OF HOST‐SPECIFIC PARASITIC FUNGI?1

Understanding of the genetic basis for susceptibility and resistance is still lacking for most aquatic host–parasite systems, for instance, for phytoplankton and their fungal parasites. Fungal parasites can have significant effects on phytoplankton populations, mainly through their ability to decimate algal host populations during epidemics. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis to study levels of genetic variation within a population of the freshwater diatom Asterionella formosa Hassall in relation to parasitism by the obligate, host‐specific, fungal parasite Zygorhizidium planktonicum Canter. The level of genetic variation within the A. formosa population in Lake Maarsseveen, The Netherlands was found to be high despite the presumed absence or very low frequency of sexual reproduction in this species, the limited gene flow, and the severity of parasite attack that would purge the population from susceptible genotypes. RAPD analysis revealed four distinct banding patterns, with 3 of 21 markers (14%) being polymorphic. In AFLP analysis, every single isolate of A. formosa showed a unique banding pattern, and 120 of the 210 AFLP markers (57%) were found to be polymorphic. Furthermore, character compatibility analysis revealed that sexual reproduction may be one of the mechanisms that generates and maintains genetic variation in the A. formosa population in Lake Maarsseveen. The presence of genetic variation in A. formosa was reflected in infection experiments, which showed that genetically different A. formosa strains differed in their susceptibility to various Z. planktonicum strains and that parasite strains differed in their ability to infect particular host strains.     

Molecular characterization of Yarrowia lipolytica and Candida zeylanoides isolated from poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.