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PubMed2006

Primer R108 performs best in the RAPD strain typing of three Aspergillus species frequently isolated from patients.

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.

Aspergillosis↗
PubMed2001

Identification and subtyping of Trichophyton mentagrophytes by random amplified polymorphic DNA.

Trichophyton mentagrophytes is one of the most common pathogens for human and animal dermatophytoses and known as a complex species with variable morphology, and ecologic and genetic backgrounds. We performed random amplified polymorphic DNA (RAPD) analysis on 43 human and 18 animal isolates of T. mentagrophytes along with other 10 anamorphic species of dermatophytes and three teleomorphic species of T. mentagrophytes. Using RAPD analysis with primer 5'-ATGGATC(G,C)(G,C)C-3' (ATGS), all T. mentagrophytes strains produced identical band patterns with those of Arthroderma vanbreuseghemii, one of the teleomorphs of T. mentagrophytes, regardless of their phenotypes. Therefore, T. mentagrophytes could be identified by RAPD analysis with primer ATGS. Using RAPD analysis with primer 5'-ATGGATCGGC-3' (ATG) on T. mentagrophytes, human isolates yielded two distinct subgroups related by their colony morphologies at the time of primary isolation from patients. Three morphologic types--cottony, powdery and persicolor--revealed identical bands whereas the granular type lacked one minor band (0.74 kbp). Animal isolates of T. mentagrophytes produced five band patterns and some of them were identical with those of human isolates. With primer 5'-GAAGGCTCCC-3' (OPAO-15), animal isolates of T. mentagrophytes showed diverse band patterns in contrast to the uniform band pattern of human isolates. These results suggest that RAPD analysis may be a useful tool to identify and subtype T. mentagrophytes complex.

Animals↗
PubMed2006

Evaluation of a modified single-enzyme amplified fragment length polymorphism (SE-AFLP) technique for subtyping Salmonella enterica serotype Enteritidis.

Salmonella enterica subsp. enterica serotype Enteritidis is not readily subtyped beyond the level of phage type (PT). Pulsed field gel electrophoresis (PFGE) is generally acknowledged to be the most discriminating typing method for Salmonella, but only a restricted variety of PFGE types has been described for S. enterica serotype Enteritidis. In the present study, a modification of the SE-AFLP typing method was used to investigate both outbreak and apparently sporadic isolates of S. enterica serotype Enteritidis belonging to different PTs and/or PFGE types. The method proved to be as discriminatory as PFGE when combined with phage typing, and provided subtyping data consistent with epidemiological information. Although the modified SE-AFLP typing method did not prove to achieve a superior discriminatory ability in resolving clusters, it has a high enough throughput for use in outbreak investigations. This method can be used in combination with other typing methods to obtain epidemiologically relevant subtyping data on S. enterica serotype Enteritidis.

Bacterial Typing Techniques↗
PubMed1997

Random amplification of polymorphic DNA is useful for the differentiation of several anthropophilic dermatophytes.

The efficacy of random amplification of polymorphic DNA (RAPD) analysis, a polymerase chain reaction (PCR)-based technology, as a tool for differentiation of several anthropophilic dermatophytes, that is, Trichophyton mentagrophytes var. interdigitale, T. rubrum and Epidermophyton floccosum, was examined. Total cellular DNA extracted by a mini-preparation method were used as template DNAs and PCRs were performed using five primers, all of which were synthesized 10-mers. All of the primers generated PCR products and their electrophoresed profiles were characteristic for each of the species. Since these anthropophilic dermatophyte species could be differentiated clearly by the RAPD analysis, this method may be useful for identification of these species. Minimal intraspecies polymorphisms were observed between the isolates of T. mentagrophytes var. interdigitale. The banding patterns were reproducible using template DNAs prepared at different times from each of stock cultures.

Arthrodermataceae↗
PubMed1996

RAPD typing in microbiology--a technical review.

Many biochemical and molecular techniques can be used for distinguishing isolates of a given bacterial species. Traditional typing techniques based on phenotypic characteristics such as serotyping are being increasingly challenged by the use of DNA-based methods. The introduction of the polymerase chain reaction (PCR) has led to typing techniques based on DNA amplification. Randomly amplified polymorphic DNA (RAPD) typing (also known as arbitrarily primed-polymerase chain reaction, APPCR) is one such technique which is being used increasingly to type micro-organisms, especially during clinical outbreaks. The application and potential problems and solutions of RAPD typing are discussed and the role of such techniques among established typing methods is addressed.

Bacterial Typing Techniques↗
PubMed2003

Isolation of Legionella anisa from multiple sites of a hospital water system: the eradication of Legionellacontamination.

For the prevention of nosocomial cases of legionellosis in the Ryukyu University Hospital neonatal wards, we examined nine shower units and a sink tap water unit for the presence of Legionella, over a 6-year period. We isolated Legionella-like organisms (LLO) from showerheads by culturing sediments from the water samples on buffered charcoal yeast extracts (BCYE). We used DNA-DNA hybridization to determine that the organisms were L. anisa. A fingerprinting technique called random amplified polymorphism DNA (RAPD) also showed that all the organisms were identical at the genome level. Replacement of the shower heads harboring colonies of L. anisa prevented further contamination. Nosocomial cases of legionellosis have not been reported from the wards during the period of this survey. This is the first description of the isolation of L. anisa from multiple sites within a hospital, and RAPD analysis suggests that these may be the spread of a single clone.

Cross Infection↗
PubMed1997

Differentiation of Arthroderma spp. by random amplification of polymorphic DNA (RAPD) and Southern hybridization.

To develop the molecular differentiation analysis of dermatophytes, we carried out RAPD and Southern hybridization analyses using genomic DNAs of six Arthroderma species, including A. fulvum, A. grubyi, A. gypseum, A. incurvatum, A. otae and A. racemosum. The RAPD analysis gave different band patterns specific to each of the six Arthroderma fungi. However, minor differences in the banding patterns were observed between the strains of plus (+) and minus (-) mating types of A. gypseum, A. fulvum and A. incurvatum. Southern blot analysis using a probe (1S) obtained from A. grubyi DNA gave specific bands only in the DNA samples of A. grubyi and A. incurvatum. On the other hand, Southern blot analysis using a probe (C3) obtained from A. otae DNA gave specific bands in all six Arthroderma species examined, and the size of the bands were specific to each species. These findings indicate that RAPD and Southern hybridization analyses are useful in the differentiation of these Arthroderma species.

Amino Acid Sequence↗
PubMed1998

Molecular typing of Malassezia species with PFGE and RAPD.

The currently recognized seven species of Malassezia all have different karyotypes which do not vary intraspecifically, except in M. furfur which displayed two different karyotypes. In contrast, random amplified polymorphic DNA (RAPD) typing showed the presence of genetic variation in all species. It is concluded that karyotype analysis is useful for species identification, and RAPD typing can be used in epidemiological investigations.

Animals↗
PubMed2005

Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD).

Trichophyton rubrum is an important cause of dermatomycoses. Molecular strain typing methods have recently been developed to address questions about epidemiology and source of relapse following treatment. This report describes the application of RAPD for molecular strain differentiation of this fungus utilizing the primers 1- (5'-d[GGTGCGGGAA]-3') and 6- (5'-d[CCCGTCAGCA]-3'). A total of five RAPD patterns were observed among 10 strains of T. rubrum, with each of the primers used. We conclude that RAPD analysis using primers 1 and 6 can be used in epidemiological studies.

DNA Primers↗
PubMed2000

Strain characterization of Candida parapsilosis fungemia by molecular typing methods.

The present study used two molecular typing methods to investigate a cluster of eight cases of Candida parapsilosis fungemia in a hospital in Rio de Janeiro, Brazil. Candida parapsilosis is an important opportunistic pathogen that is frequently involved in outbreaks of nosocomial fungemia. Identification of a common source of infection and determination of genetic relatedness among the strains involved in outbreaks are important for infection control. Candida parapsilosis strains were isolated from the bloodstream of patients housed in an intensive-care unit (n=5) and in individual rooms (n=3). An additional strain of Candida parapsilosis was isolated from a hyperalimentation infusion flask, which was implicated by molecular typing to be the source of infection. All strains were identified using morphological and biochemical methods. The genetic relationship between patients' strains and the hyperalimentation infusion strain was assessed by electrophoretic karyotype (EK) analysis and random amplification of polymorphic DNA (RAPD). Both methods resulted in patterns that allowed differentiation of the isolates. Candida parapsilosis fungemia, in three of the eight patients, resulted from a common source of infection, as demonstrated by molecular typing methods. Image analysis of EK patterns indicated that these strains were closest to Candida parapsilosis Group II, a grouping that is a less frequent clinical isolate than the major Group I strains.

Candida↗
PubMed1999

Diversity among wild type and vaccination strains of Trichophyton verrucosum investigated using random amplified polymorphic DNA analysis.

We initially tested 20 primers for their ability to amplify genomic DNA of Trichophyton verrucosum using RAPD. Six of these were selected for further study aimed at discrimination of wild type and vaccination strains of T. verrucosum. The results indicate that RAPD successfully distinguished all strains included in the study. In addition, results of corrected cluster analysis were consistent with the fact that the avirulent vaccination strains (T. verrucosum TV-M9 and T. verrucosum TV-M-130) were prepared by ultraviolet (UV) light induced mutagenesis of the standard wild type strain T. verrucosum Strádznice. No marker for a/virulence was detected. These outcomes suggest new possibilities for epidemiological analyses, for discrimination among different vaccination strains and studies of fungal population in vaccinated/infected hosts.

Animals↗
PubMed2002

Limited genetic diversity of Candida albicans in fecal flora of healthy volunteers and inpatients: a proposed basis for strain homogeneity in clinical isolates.

Molecular analysis of Candida albicans isolates from individual patients often yields a single strain at multiple sites. Whether this strain-limitation is due to virulence factors favoring the invasive strain or to lack of genetic diversity in the gastrointestinal reservoir is uncertain. We elected to study C. albicans genotypes in the fecal flora among healthy volunteers and inpatients. Self-obtained stool swabs or stool samples were cultured on inhibitory mold agar. From each subject with C. albicans, nine colonies were randomly selected, individually propagated, and typed utilizing random amplified polymorphic DNA. Colonies were considered identical (all bands matched), related variants (one to three unique bands), or distinct strains (more than three unique bands). Analysis showed a single clone in 33/43 (76.7%) volunteers and 6/18 (33.3%) inpatients (P = 0.018), two to four related variants in eight (18.6%) volunteers and 10 (55.6%) inpatients, and two distinct strains in two volunteers (4.6%) and two inpatients (11.1%). Strain variation was more common in females (33.5 versus 5.6%; P = 0.04) and tended to increase with age (r = 0.245, P = 0.06). These findings illustrate that most healthy subjects harbor a single strain of C. albicans in the fecal flora. This strain may undergo genetic evolution leading to minor clonal variations. The mechanisms for strain selection, maintenance and possible evolution remain to be delineated.

Candida albicans↗
PubMed2001

Molecular typing of Candida isolates from patients hospitalized in an intensive care unit.

OBJECTIVES: The aim of our study was the molecular typing of 40 clinical isolates of Candida spp. obtained from patients with burns or trauma hospitalized in the intensive care unit of a general hospital. METHODS: Isolates were recovered from blood, deep trauma, urine, sputum or from environment within a short period of time (4 months). The yeasts were identified using commercial yeast identification kits as C. albicans (17 isolates), C. tropicalis (16 isolates) and C. parapsilosis (10 isolates). The epidemiological relation of the isolates was tested with the Random Amplified Polymorphic DNA assay using three or four arbitrary chosen primers. RESULTS: All C. albicans isolates presented distinct RAPD profiles, C. tropicalis isolates presented both the same and distinct RAPD patterns and the C. parapsilosis isolates presented the same RAPD pattern. All the environmental isolates were identified as C. parapsilosis and they had the same RAPD pattern as C. parapsilosis clinical isolates. Candida parapsilosis delineation was confirmed with PFGE. CONCLUSIONS: The colonization/infection with C. albicans was endogenous, the C. tropicalis colonization/infection was both endogenous and exogenous, and the C. parapsilosis colonization/infection had an environmental origin.

Burns↗
PubMed1998

Aspergillus fumigatus strains recovered from immunocompromised patients (ICP): subtyping of strains by RAPD analysis.

Invasive pulmonary aspergillosis in immunocompromised patients (ICP) is the second most frequent opportunistic fungal infection. The causative organism includes 16 species of Aspergillus, of which A. fumigatus dominates the ubiquitous incidence of invasive or allergic broncho-pulmonary aspergillosis (ABPA). The definitive diagnosis of invasive aspergillosis is difficult. We have analyzed 24 strains of A. fumigatus recovered from ICP using the RAPD technique. The profiles generated with the 20 primers tested were mostly unique. These results may have a profound impact on the management of aspergillosis, especially in the ICP.

Aspergillosis↗
PubMed2002

Genotyping study of Scedosporium apiospermum isolates from patients with cystic fibrosis.

Usually a saprophyte, Scedosporium apiospermum often colonizes the respiratory tracts of patients with cystic fibrosis (CF). In order to improve our understanding of the molecular epidemiology of the airway colonization, 129 sequential and multiple isolates collected from January 1998 to March 1999 from nine CF patients monitored in three hospitals in France were typed by random amplification of polymorphic DNA with primers GC70, UBC-701, and UBC-703. Among these primers, UBC-703 was the most discriminating, allowing the differentiation of 14 genotypes. Combining the results obtained with this three-primer set resulted in the differentiation of 16 genotypes. No common genotype was found among the different patients, and no clustering according to geographic origin of the isolates was seen. In addition, five of the patients were colonized by a single genotype. The others usually exhibited a predominant genotype accompanied by one or two others, which were found occasionally and were genetically close to the predominant genotype. Thus, our study demonstrates the persistence of the fungus despite antifungal treatments and therefore reinforces the need for the development of new antifungals that are more efficient against this species.

Adolescent↗
PubMed2002

Utility of a pre-optimized kit for random amplified polymorphic DNA in typing Candida albicans.

The utility of a pre-optimized kit for random amplified polymorphic DNA (RAPD) was assessed in typing diverse strains of Candida albicans from epidemiologically unrelated inpatients (interpatient analysis) and in detecting clonal variations that maybe present within individual patient isolates (intrapatient analysis). Stool samples from inpatients were cultured on Inhibitory Mold agar. Nine individual colonies from all patients with > or =9 colonies of C. albicans (n = 18) were selected, frozen, and karyotyped using CHEF genomic DNA plug kits and CHEF-DRIII. Each of the selected colonies was then analyzed by RAPD, utilizing the selected kit, with 6 primers. Interpatient analysis revealed 9 karyotypes and 17 RAPD composites. RAPD discrimination was significantly better (p < 0.001). Intrapatient analysis revealed 34 (21%) and 33 (20.4%) variants among 162 colonies tested by RAPD and karyotyping, respectively. The results were discordant in 25 variants, all with differences of 1-3 bands. These results illustrate that this pre-optimized kit for RAPD provides excellent discrimination of genetically unrelated strains. Its performance in delineating subtle clonal differences was comparable with karyotyping; both methods failed to detect all minor genetic variations. The ease of use and quick turnaround time of this kit offer a practical and reliable method for typing diverse strains of C. albicans, but may be inadequate for assessing microevolution.

Candida albicans↗
PubMed1995

Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider).

We have applied the recently developed technique of random amplified polymorphic DNA (RAPD) for the discrimination between two jojoba clones at the genomic level. Among a set of 30 primers tested, a simple reproducible pattern with three distinct fragments for clone D and two distinct fragments for clone E was obtained with primer OPB08. Since RAPD products are the results of arbitrarily priming events and because a given primer can amplify a number of non-homologous sequences, we wondered whether or not RAPD bands, even those of similar size, were derived from different loci in the two clones. To answer this question, two complementary approaches were used: i) cloning and sequencing of the amplification products from clone E; and ii) complementary Southern analysis of RAPD gels using cloned or amplified fragments (directly recovered from agarose gels) as RFLP probes. The data reported here show that the RAPD reaction generates multiple amplified fragments. Some fragments, although resolved as a single band on agarose gels, contain different DNA species of the same size. Furthermore, it appears that the cloned RAPD products of known sequence that do not target repetitive DNA can be used as hybridization probes in RFLP to detect a polymorphism among individuals.

Base Sequence↗