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PubMed2002

Identification of Legionella species by ribotyping and other molecular methods.

There are currently more than 40 species of Legionella and the identification of most of them by standard methods is often technically difficult. The aim of this study was to use a ribotyping method with endonuclease HindIII and a probe consisting of a set of five oligonucleotides (referred to as OligoMix5). A total of 123 strains, including 78 type or reference strains corresponding to 44 species, eight clinical and 37 environmental isolates were tested. The usefulness of the method was demonstrated for the identification at the species level of all of the 123 Legionella isolates tested, with each species showing a specific profile. Among the 15 serogroups of Legionella pneumophila, eight patterns were obtained. For the 45 field strains, the randomly amplified polymorphic DNA (RAPD) technique and intergenic 16S-23S ribosomal spacer PCR analysis (ITS 16-23S) were also used. Altogether, these three methods allowed the identification of all of strains tested. However, ribotyping has proven to be more effective than the other methods.

Environmental Microbiology↗

PubMed2003

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique.

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.

Basidiomycota↗

PubMed2001

Genetic heterogeneity of Mycoplasma hominis clinical isolates detected during observation of patients with recurrent urogenital inflammation.

A rapid reproducible effective method for molecular typing of Mycoplasma hominis strains based on random amplified polymorphic DNA (RAPD) technique was developed. RAPD detected genetic heterogeneity of genomes of Mycoplasma hominis clinical isolates and showed changes in the genomes of Mycoplasma hominis clinical isolates from patients with chronic infection.

Adolescent↗

PubMed2005

Relationship among Lepista species determined by CAPS and RAPD.

To determine the relationship of Australian members of the genus Lepista with those from other parts of the world, genetic variation of isolates representing 27 accessions was assayed by cleaved amplified polymorphic sequences (CAPS) and random amplified polymorphic DNA (RAPD). CAPS and RAPD identified eight and seven groups, respectively. CAPS Groups 1 and 2 and RAPD Group 1 consisted of French and Australian accessions classified as morphospecies L. nuda. CAPS Group 3 and RAPD Groups 2 and 2A consisted of mostly Australian isolates identified as L. sordida var. sordida or L. sordida var. umbonata. Isolates earlier identified as morphospecies L. sp. were also placed in CAPS Group 3 and RAPD Group 2A indicating that these isolates are L. sordida var. sordida. In addition, three smaller groups were distinguished. A French isolate of L. sordida var. sordida was placed in distinctly separate CAPS and RAPD groups to Australian L. sordida var. sordida Groups 4,4 respectively. A French isolate of L. sordida var. aianthina was placed in CAPS and RAPD Groups 3,3. An accession of L. saeva was placed in CAPS Group 6 and RAPD Group 5, separate from other isolates. RAPD Groups 6 and 7 consist, respectively, of Greek and American accessions of L. nuda that were only distantly related to the Australian and French accessions of this morphospecies: CAPS also separated these isolates from each other and from all other isolates. The data suggest that the classification of morphospecies and varieties within Lepista cannot be determined on the basis of morphology alone. The Greek and American accessions of L. nuda are separated from the French and Australian accessions and may not be L. nuda. Similarly, the Australian accessions currently classified as L. sordida var. sordida together with the accessions of L. sordida var. umbonata are distinct from the French accessions of L. sordida var. sordida suggesting that Australian isolates may represent a new species or variety. Further studies using a combination of morphological and genetic characterization are required to provide a more accurate understanding of Lepista species.

Agaricales↗

PubMed2005

Genetic structure of the Pleurotus eryngii species-complex.

Assessment of genetic and phenotypic diversity is necessary to confidently distinguish genotypes of Pleurotus eryngii when seeking traits of interest and to identify strains with high yield potential. We studied 154 strains from Italy for quantitative (shape, size and yield), qualitative (colour, malformations and growing behaviour), and molecular (RAPD and minisatellite) traits. This population consisted of isolates mostly belonging to P. eryngii var. eryngii, var. ferulae or var. nebrodensis, from different regions of Italy. A replicated cultivation trial, with three blocks and three replicates for each strain within the block, was used as an experimental design to calculate trait estimates. Significant differences were observed between strains for basidiome number and weight, while no significant differentiation for quantitative morphological traits was observed between geographical origins and taxonomic groups. Qualitative morphological traits were efficient in differentiating isolates of P. eryngii var. nebrodensis. On average, yield per strain (basidiome weight) was correlated more with basidiome number than with size. The most stable yield traits were basidiome number and weight per strain. An average heritability of 0.31 was estimated for yield related traits. A significant difference between var. ferulae and var. eryngii populations was detected for basidiome production measured as 'average harvest time'. Molecular markers showed a high level of heterogeneity within populations and a low, but significant, degree of differentiation among populations defined a priori. No population-specific marker was detected and the differential pattern of variation between vars. ferulae and eryngii was due to frequency-dependent alleles. The nebrodensis type was more differentiated from var. eryngii than from var. ferulae using either molecular or qualitative morphological traits.

Genetic Markers↗

PubMed2005

Use of random amplified microsatellites to type isolates from an outbreak of nosocomial aspergillosis in a general medical ward.

Numerous patients were diagnosed with aspergillosis in a nosocomial outbreak caused by Aspergillus fumigatus and Aspergillus flavus. Thirty-three isolates of the former and 28 isolates of the latter were collected from the hospital environment and from the patients and studied for genetic relatedness by random amplified microsatellites (RAMS) analysis, in which two polymorphic regions were tested. Twenty-eight genotypes of A. fumigatus and 23 genotypes of A. flavus were identified. Four patients were infected by two isolates with the same genotype as the environmental isolates. One clinical genotype was shared by three patients and another was shared by two patients. We found that RAMS was useful for fingerprinting Aspergillus spp.

Aged↗

PubMed1999

Diphtheria in the Republic of Georgia: use of molecular typing techniques for characterization of Corynebacterium diphtheriae strains.

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates.

Bacterial Typing Techniques↗

PubMed1999

Value of different methods for the characterisation of Aspergillus terreus strains.

To evaluate different methods for strain differentiation, 10 isolates of Aspergillus terreus from Germany and two epidemiologically unrelated strains were investigated. The sources of the isolates were patients with cystic fibrosis (4), immunosuppression (2), otitis externa (2), sinusitis (1) and endocarditis (1). Environmental isolates were obtained from a contaminated cell culture and from soil. The isolates did not differ in their macroscopic and microscopic morphology, in their protein patterns analysed by SDS-PAGE and in their susceptibility to amphotericin B and itraconazole. The RFLP analysis of total genomic DNA digested by EcoRI resulted in patterns that were too faint for interpretation. However, after hybridisation of the digested DNA with a short DNA probe of repetitive sequence, six different patterns were found. Based on the patterns of the randomly amplified polymorphic DNA (RAPD) with three primers, nine different genotypes were discriminate. RAPD patterns discriminated the epidemiologically unrelated reference strains (endocarditis isolate from Thailand, soil isolate from the USA) and the isolates from Germany. It is concluded that, in contrast to the phenotypic methods, the analysis of RAPD patterns is useful for strain differentiation of A. terreus.

Amphotericin B↗

PubMed2000

The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application.

The random amplified polymorphic DNA (RAPD) technique was applied to settle a lawsuit involving unauthorized commercialization of a patented strawberry variety of high economical relevance ('Marmolada'). Because of economical involvements, the molecular approach was added to the more traditional morphological examination in a double-blind test. All plants belonging to the patented variety were unambiguously identified (13 plants among a total of 31 plants examined). The results were accepted as evidence in the court. This study confirms that the RAPD technique is especially suitable for identification of asexually reproduced plant varieties for forensic or agricultural purposes.

Fruit↗

PubMed2004

Use of molecular methods in identification of Candida species and evaluation of fluconazole resistance.

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

Antifungal Agents↗

PubMed2003

[Molecular genetic diagnostics of some Daphnia species (Crustacea, Cladocera) from the Volga river].

Population genetic structure of the widespread Daphnia species from Kuibyshev and Saratov reservoirs was examined by use of RAPD-PCR technique with four mitochondrial DNA (16S rRNA) specific primers. One of the examined Daphnia clones from the Volga region appeared to be conspecific to the North American Daphnia galeata, while another clone was most likely a hybrid between D. galeata and D. cucullata.

Animals↗

PubMed2000

The ins and outs of DNA fingerprinting the infectious fungi.

DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies.

DNA Fingerprinting↗

PubMed2002

Intra-strain variability of Cryptococcus neoformans can be detected on phloxin B medium.

A method was devised for easy detection of intra-strain variability of the human pathogenic yeast Cryptococcus neoformans. Cultivation of strains on a medium containing Phloxin B resulted in different coloured colonies. Generally, colonies were either pink or red; however there were also several colony-colour segregant in which both colours could be observed. A number of these segregants were isolated and analysed. Virulence factors such as the cell and capsule sizes were measured; further temperature sensitivity, growth rates, mating-types and melanin production were also studied. Segregants were examined by random amplified polymorphic DNA (RAPD) fingerprinting and electrophoretic karyotyping by pulsed-field gel electrophoresis (CHEF). They showed both phenotypic and genotypic differences. The main differences appeared in phenotypic characters and RAPD patterns; while the chromosomal patterns remained unchanged. Reversion frequency analysis revealed that the reason for this segregation could be due to phenotypic switching. The physiological reason for the colour changes was also investigated and was attributed to the differential ability of the cells to accumulate Phloxin B either into their capsules or into their cells. The method described here is potentially applicable for the detection of strain heterogeneity in both basic and clinical microbiology laboratories.

Cryptococcus neoformans↗

PubMed1995

Genome fingerprinting of the silkworm, Bombyx mori, using random arbitrary primers.

The random amplified polymorphic DNA (RAPD) technique was used to study DNA profiling of thirteen silkworm genotypes. The genotypes included six diapausing and seven nondiapausing varieties that represent a high degree of divergence with respect to geographic origin, and morphological, qualitative, quantitative and biochemical characters. Two hundred sixteen amplified products were generated using 40 random primers. Genotype-specific amplification products were identified. Amplification products specific to diapausing genotypes were also identified. Segregation of the RAPD marker was analyzed in a backcross population and found to be inherited as dominant Mendelian traits. Based on pairwise comparison of amplified products, the genetic similarity was performed by a hierarchical clustering technique. Silkworm genotypes were clustered into two groups, one consisting of six diapausing and the other of seven nondiapausing genotypes. The results of our study suggest that the RAPD technique could be used as a powerful tool to generate genetic markers that are linked to traits of interest in the silkworm.

Animals↗

PubMed2002

Differentiation of nocardia species by PCR-randomly amplified polymorphic DNA fingerprinting.

Representatives of fifteen validly described and three non-validly described species of Nocardia were assigned to nineteen groups based on an optimised PCR-randomly amplified polymorphic DNA fingerprinting technique. Species specific banding patterns were recognised for the representatives of N. brasiliensis, N. crassostreae, N. farcinica, N. otitidiscaviarum and N. seriola. Unique banding patterns were also seen for the type strains of N. brevicatena, N. carnea, N. salmonicida, N. uniformis and N. vaccinii, and for the single representatives of "N. fusca", "N. pseudosporangifera", and "N. violaceofusca". More than one banding pattern was detected for the N. asteroides, N. flavorosea, N. nova, N. pseudobrasiliensis and N. transvalensis strains though in the case of the representative strains of N. nova and N. transvalensis the patterns were similar for each of these species. The results are in line with current trends in nocardial systematics thereby indicating that PCR-randomly amplified polymorphic DNA fingerprinting provides valuable data for the classification and identification of pathogenic nocardiae to the species level.

DNA Fingerprinting↗

PubMed2004

Reservoir of Candida albicans infection in a vascular bypass graft demonstrates a stable karyotype over six months.

We retrospectively analyzed five Candida albicans isolates from two infection episodes in a single patient 6 months apart. Using contour-clamped homogeneous field electrophoresis (CHEF), random amplified polymorphic DNA (RAPD) fingerprinting, and restriction fragment length polymorphism (RFLP) complex probe 27A as means of molecular typing, we demonstrate an unvarying genotype amongst the infection-causing C. albicans strains. Several months later, the patient yielded C. glabrata in a yeast survey of oral and rectal sites. The preponderance of C. glabrata and lack of C. albicans isolated from normal flora sites suggests that this patient harbored the prior C. albicans bloodstream isolate on a Gore-Tex graft for 6 months prior to the second episode of fungemia.

Blood↗

PubMed2006

Genetic and geographic polymorphism of cultivated tobaccos (Nicotiana tabacum) in Turkey.

Nicotiana tabacum (2n = 48) is a natural amphidiploid and shows a distribution over a geographical area in eastern anatolia. Random amplified polymorphic DNA (RAPD) technique was used to evaluate both genetic diversity among 21 primitive tobacco accessions comparing flue cure virginia genotype (FCV) and their geographical polymorphism as a source of genetic variations for breeding programs. Only 13 of all the 60 random primers used in RAPD showed polymorphism acceptable for characterization of these accessions. Totally 118 RAPD fragments were generated from thirteen decamer primer and sixtyfour of them were found polymorphic (54.2%). Mus and FCV showed the smallest genetic distance among accessions cultivated in the eastern anatolia. These results shows that the RAPD assay is a powerful approach for identifying genetic and geographic polymorphism.

DNA, Plant↗

PubMed2006

Genotypic relatedness of yeasts in thrush and denture stomatitis.

BACKGROUND/AIM: Candida is an opportunistic pathogen. Understanding its genetic characters might increase our understanding of the pathogenesis of candidosis. We examined the genetic relationships of yeasts from the most common forms of oral candidosis: thrush and denture stomatitis. METHODS: Yeasts were sampled from palate, buccal mucosa, gingival sulci/periodontal pockets and/or denture fitting surface of 19 thrush patients and 22 denture stomatitis patients. Random amplified polymorphic DNA and the Dendron computer-assisted program were used to determine the genotypic relatedness of the yeasts. RESULTS: A dendrogram generated from 105 thrush isolates had similarity coefficients (S(AB)) ranging from 0.58 to 1 with four clusters derived at S(AB) 68%. Another dendrogram was generated from 91 isolates from denture stomatitis, with S(AB) ranging from 0.59 to 1. Three clusters were established at S(AB) 71%. In a composite dendrogram incorporating the thrush and denture stomatitis data and orally healthy data compiled from a previous study, five genotypic clusters were generated at S(AB) 68%. Cluster II, the most dominant, comprised isolates from thrush, denture stomatitis and healthy conditions, while clusters III and IV contained yeasts mainly from thrush. CONCLUSIONS: Palatal yeast carriage was significantly increased in thrush and denture stomatitis, also after radiation, chemotherapy and denture wearing. The buccal mucosa was favorable for yeast colonization regardless of oral condition. Yeasts in thrush were more diverse than in conditions of oral health. The common clone (II) of infecting yeasts and commensals suggested that commensals could induce thrush and denture stomatitis, whereas the unique clones in thrush (III, IV) might have been established through strain replacement or maintenance with minor genetic variation.

Adolescent↗