PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Random Amplified Polymorphic DNA Technique”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources

An endophyte of the tropical forage grass Brachiaria brizantha: isolating, identifying, and characterizing the fungus, and determining its antimycotic properties.

Brachiaria, predominantly an African genus, contains species, such as B. brizantha, an apomictic C4 grass, that are commercially important forage grasses in tropical America, where they now cover about 55 million hectares. From B. brizantha accession CIAT 6780, we isolated an endophytic fungus that may be economically significant. The fungus was identified as Acremonium implicatum (J. Gilman & E.V. Abott). 18S rDNA and ITS rDNA sequences were used to characterize isolates of the endophyte, and showed that they belonged to the Acremonium genus, being close to A. strictum and A. kiliense. Using the random amplified polymorphic DNA (RAPD) technique, involving arbitrary primers of 10 bases, we showed that the isolates were highly similar to each other. Antiserum produced from a monoconidial culture of A. implicatum isolated from B. brizantha 6780, differentiated the isolates consistently in line with the DNA data. When we compared endophyte-free with endophyte-infected B. brizantha CIAT 6780 plants, both artificially inoculated with the pathogenic Drechslera fungus, we found that the endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte.

Acremonium↗

A new approach for the identification and the diagnosis of Eimeria media parasite of the rabbit.

In this work, we described a new approach for the isolation of a species-specific probe for the Eimeria media parasite of the rabbit based on the use of the random amplified polymorphic DNA (RAPD) technique. A specific fragment of 800 bp of the studied species was isolated after RAPD and then cloned and DIG-radiolabeled. After dot-blotting, we observed that this probe was specific for E. media. Sequencing of the 3' and 5' ends of this probe enabled the determination of two primers that could be used in a PCR reaction. The amplified product of 750 bp was specific E. media. The use of these primers and of our probe allowed the detection of a very small number of oocysts. With a new protocol of DNA purification, 10 purified oocysts were detected by PCR. The efficiency of the amplification was not changed when two species were mixed. The threshold of detection of oocysts in fecal matter was equal to 30.

Animals↗

Combined used of RAPD and touchdown PCR for epidemiological studies of Aspergillus fumigatus.

INTRODUCTION: Aspergillus fumigatus is a filamentous fungus that acts as an opportunistic pathogen and has emerged as a major problem in immunosuppressed patients. Nosocomial outbreaks of aspergillosis are becoming more frequent, but their identification and epidemiological characterization is slow and difficult. OBJECTIVE: Description of a fast, sensitive, specific method to identify and fingerprint A. fumigatus using methodology available in clinical laboratories. METHODS: We studied several strains of A. fumigatus isolated from patients with invasive aspergillosis (n = 4), the hospital environment (n = 5) and reference cultures (n = 1), as well as other close phylogenetic fungal species from patients (n = 1), hospital environment (n = 6) and reference cultures (n = 1). A. fumigatus was identified by both touchdown PCR and conventional phenotyping methods. Genotyping was performed with random amplification of polymorphic DNA (RAPD) analysis, comparing the results from two primers (OPZ-19 and R-108) and different amplification protocols with regard to band resolution and reproducibility. RESULTS: Touchdown PCR and phenotype results were identical. Best RAPD results were obtained with the R-108 primer and considerably longer ramp times between annealing and extension. CONCLUSION: RAPD analysis is a fast, reliable tool for DNA fingerprinting. Patterns may be easier to repeat and interpret when longer ramp times are used. Touchdown PCR combined with RAPD analysis is a sensitive, accurate method for managing clinical outbreaks of Aspergillus fumigatus.

Aspergillosis↗

Characterization of gibberellin producing strains of Fusarium moniliforme based on DNA polymorphism.

The gibberellins are one of the major groups of growth promoting hormones and are secondary metabolites of the fungus Fusarium moniliforme (Perfect stage: Gibberella fujikuroi). Sixteen strains of Fusarium from different geographical regions and different hosts were analysed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). Range of gibberellin production varied between 28.9 to 600.0 mg g(-1) dry weight of mycelium in different strains of Fusarium. RAPD analysis showed completely different pattern between high, moderate and low producing strains. High producers formed nearly identical RAPD patterns, whereas the low and moderate producers gave heterologous amplification patterns. Since Fusarium pallidoroseum was in another group, it was possible to distinguish between different species of the genus Fusarium by RAPD. These investigations may find an application in the diagnosis of unknown Fusarium species and in distinguishing isolates of Gibberella fujikuroi within the section of Liseola.

DNA, Fungal↗

Comparative analysis of genetic variability among Candida albicans isolates from different geographic locales by three genotypic methods.

The objective of the present study was to conduct a comparative genotypic analysis of Candida albicans isolates from the United States, Europe, and Southeast Asia to determine whether differences between isolates might be associated with geographic locations. The genotypes of 86 unrelated isolates of C. albicans (from the United States and Europe) and 26 isolates from Singapore were examined by three DNA typing methods. Computer-assisted methods were used to analyze the gel patterns for all isolates. A dendrogram based on the overall similarity of the patterns obtained by restriction endonuclease analysis (REA) with EcoRI clustered the U.S. and European isolates into two major groups (groups A and B). The Singaporean isolates demonstrated unique REA profiles, with nine isolates having both or neither of the REA-characteristic 3.7- and 4.2-kb bands present in groups A and B. By REA profiles, the Singaporean isolates were related to each other with similarity values (S(AB)s) of > 0.80, but only one isolate mixed with the U.S. and European isolates at this S(AB) (an arbitrary threshold for genetic similarity). Randomly amplified polymorphic DNA (RAPD) analysis generated DNA profiles that clustered the C. albicans isolates into approximately the same number of distinct typing groups as REA. However, isolates identical to each other by REA were generally different from each other by RAPD analysis. In a composite dendrogram prepared from the results obtained by RAPD analysis, the isolates from the United States and Europe clustered in major groups with S(AB)s of > 0.85, while Singaporean isolates connected to these clusters at S(AB)s of > or = 0.75. Pulsed-field gel electrophoresis was less discriminatory, discerning about one-third as many distinct subtypes as REA or RAPD analysis; the Singaporean isolates were distributed randomly with the U.S. and European isolates. These results suggest that a high degree of genetic diversity exists between C. albicans isolates from Southeast Asia and those from the United States and Europe.

Candida albicans↗

Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolated from Amarone wine. Diversity of Saccharomyces strains from Amarone wine.

Individual yeast strains belonging to the Saccharomyces sensu stricto complex were isolated from Amarone wine produced in four cellars of the Valpolicella area (Italy) and characterized by conventional physiological tests and by RAPD-PCR and mtDNA restriction assays. Thirteen out of 20 strains were classified as Saccharomyces cerevisiae (ex S. cerevisiae p.r. cerevisiae and p.r. bayanus) and the remaining as Saccharomyces bayanus (ex S. cerevisiae p.r. uvarum). RAPD-PCR method proved to be a fast and reliable tool for identification of Saccharomyces sensu stricto strains and also gave intraspecific differentiation. Restriction analysis of mtDNA permitted to distinguish S. cerevisiae and S. bayanus species and to discern polymorphism among S. cerevisiae isolates. The assessment of the phenotypic diversity within the isolates by gas-chromatographic analysis of secondary fermentation products was explored. Small quantities of isobutanol were produced by most of the strains and higher amounts by some S. cerevisiae strains with phenotypes Gal- and Mel-; all S. bayanus strains produced low amounts of amilyc alcohols. From this study it appears that each winery owns particular strains, with different genetic and biochemical characteristics, selected by specific environmental pressures during the Amarone winemaking process carried out at low temperature in presence of high sugar content.

Cluster Analysis↗

Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD).

Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts.

Ascomycota↗

Different portions of the maize root system host Burkholderia cepacia populations with different degrees of genetic polymorphism.

In order to acquire a better understanding of the spatial and temporal variations of genetic diversity of Burkholderia cepacia populations in the rhizosphere of Zea mays, 161 strains were isolated from three portions of the maize root system at different soil depths and at three distinct plant growth stages. The genetic diversity among B. cepacia isolates was analysed by means of the random amplified polymorphic DNA (RAPD) technique. A number of diversity indices (richness, Shannon diversity, evenness and mean genetic distance) were calculated for each bacterial population isolated from the different root system portions. Moreover, the analysis of molecular variance (AMOVA) method was applied to estimate the genetic differences among the various bacterial populations. Our results showed that, in young plants, B. cepacia colonized preferentially the upper part of the root system, whereas in mature plants, B. cepacia was mostly recovered from the terminal part of the root system. This uneven distribution of B. cepacia cells among different root system portions partially reflected marked genetic differences among the B. cepacia populations isolated along maize roots on three distinct sampling occasions. In fact, all the diversity indices calculated indicated that genetic diversity increased during plant development and that the highest diversity values were found in mature maize plants, in particular in the middle and terminal portions of the root system. Moreover, the analysis of RAPD patterns by means of the AMOVA method revealed highly significant divergences in the degree of genetic polymorphism among the various B. cepacia populations.

Burkholderia cepacia↗

Comparison of cultural methods for the identification and molecular investigation of yeasts from sourdoughs for Italian sweet baked products.

Twenty-five yeast strains isolated from sourdough samples for Panettone, Pandoro and Cornetto brioche manufactured by eight different bakeries in northern Italy were characterised. Classification was performed by the simplified identification method (SIM), Kurtzman and Fell's identification protocol, the API system from bioMérieux (France) and the MicroLog system from Biolog (USA). Genetic diversity was investigated by randomly amplified polymorphic DNA fingerprinting and mitochondrial-DNA restriction enzyme analysis. Sequences of the internal transcribed spacers between 18S and 26S rDNA genes were analysed. Candida humilis was the predominant species (56% of isolates), whereas the remaining strains (44%) were related to the Saccharomyces cerevisiae sensu stricto group. Identification systems based on phenotypic analysis proved to be unreliable to identify yeasts from sourdough. Either RAPD-PCR or mtDNA restriction analysis showed to be suitable for the identification of species, but could not be used to differentiate among the isolates at the strain level. Sequencing of the ITS region permitted a consistent classification of the sourdough yeasts.

Bread↗

In vitro assessment of DNA damage after short- and long-term exposure to benzo(a)pyrene using RAPD and the RTG-2 fish cell line.

Genotoxins present in the aquatic environment are often associated with the decline or disappearance of many wild populations. The hazard assessment of chemicals requires sensitive and specific tests to study the genotoxic effects in order to establish the maximum allowable chemical concentrations prior to the release to the environment. We have previously shown that an established fish cell line (RTG-2) together with the random amplified polymorphic DNA (RAPD) technique, can be used to detect alterations in the DNA caused by direct acting genotoxins. The current study takes this a step further and examines in the same system the effect of a pro-mutagen benzo(a)pyrene (B(a)P) at different concentrations (0.05, 0.1, and 0.5 microg/ml) and at different exposure periods (1, 2, 3, 15, and 30 days). After comparing DNA fingerprints from control and exposed cells, both qualitative and quantitative analysis show an increase in the instability in the DNA fingerprint of exposed cells over a time- and concentration-dependent manner. At the higher concentration (0.5 microg/ml) three out the four primers showed altered bands after 1 day of exposure, while after 3 days all used primers showed an altered pattern. At the lower concentration of B(a)P (0.05 microg/ml) the appearance of new bands was observed with a 100% level of reproducibility after 30 days of exposure suggesting an inheritance of the altered DNA. We conclude that this in vitro system is useful to evaluate genotoxic effects, both after acute and chronic exposures and of direct and non-direct acting genotoxins. Cultured cells can be considered as genetically homogenous populations. Therefore, in vitro systems permits us to undertake mechanistic studies avoiding the interference of polymorphisms inherent in the in vivo systems. Furthermore, the RTG-2 fish cell line combined with a RAPD assay could be used in studies of hazard identification in risk assessment protocols of chemicals.

Animals↗

Genetic structure of sylvatic, peridomestic and domestic populations of Triatoma dimidiata (Hemiptera: Reduviidae) from an endemic zone of Boyaca, Colombia.

Randomly amplified polymorphic DNA (RAPD) technique was used to study the genetic structure of sylvatic, peridomestic and domestic populations of Triatoma dimidiata. The genetic flow among them was calculated to establish the epidemiological risk of non-domiciliated populations in the transmission of Chagas disease in an endemic area of Boyaca, Colombia. A total of 83 adult specimens were studied: 26 sylvatic, 27 peridomestic and 30 domestic insects. Wright's Fst was 0.071 and the effective migration rate (Nm) 3.3, suggestive of low genetic differentiation and a movement of at least three insects per generation. The calculated percentage of polymorphic loci was 99%, confirming a large average heterozygosity due to a permanent contact between insects of the three populations. These results imply that non-domiciliated populations of T. dimidiata represent an epidemiological risk in the transmission of Chagas disease owing to the fact that they can colonize human dwellings. Close surveillance of non-strictly domiciliated species of triatomines such as T. dimidiata should entail not only the domicile but also the peridomicile and should include control programs of animal reservoirs. Houses enhancement, educational programs, surveillance of reinfestation and of individuals at risk of infection should be priorities in the control policies in endemic regions such as Boavita, Boyaca.

Animals↗

Genetic divergence among geographical populations of the migratory locust in China.

The random amplified polymorphic DNA (RAPD) technique was used to examine genetic divergence and interrelations of 11 geographical populations of the migratory locust in China, and the role of spatial separation in the population differentiations. AMOVA analysis of genetic variations in all the populations indicated greater within- (79.55%) than among-population variability (20.45%), and that there were significant differentiations among the populations; 11 populations were divided into four regional groups, with significantly greater variability within (82.99%) than among the groups (17.01%), and there existed apparent regional differentiations. Paired comparisons showed significantly greater variability within- than between-groups, indicating significant differentiations between populations of different regional groups. Of all the pairwise comparisons, Hainan and Tibetan groups displayed the greatest differentiation, with the difference between the two groups being seven folds of that between populations within the groups; the least differentiations were exhibited between the groups of Hainan, Xinjiang, and Inner Mongolia, with the differences between groups being only half of the differences between populations within the groups. Mantel tests of the genetic and spatial distances showed that the two matrices were significantly correlated (p<0.01), indicating that the geographical isolation played an important role in the differentiations of the geographical populations of the migratory locusts. Cluster analysis divided all populations into four major groups: Xinjiang and Inner Mongolia group, the Great Plains of North China (the Yellow River and Huai River Plains) group, Hainan group, and Tibet group. Principal component analysis (PCA) supported the division of populations based on the cluster analysis. However, analysis of individuals clustered the locusts into five populations: Xinjiang and Inner Mongolia, Hami in Xinjiang, the Great Plains of North China, Hainan, and Tibet. The locust populations in eastern China displayed apparently continous and gradient variations; as such authors consider that there were no necessity and valid reasons for further division of subspecies. The subspecific status for the main geographical populations of the migratory locusts in China was discussed.

Animal Migration↗

[RAPD analysis on different isolates of Trichomonas vaginalis].

OBJECTIVE: To study genetic polymorphism of DNA on seven isolates of Trichomonas vaginalis. METHODS: The random amplified polymorphic DNA (RAPD) technique was performed to amplify genomic DNA of the seven T. vaginalis isolates, including Beijing 1, Beijing 2, Chengde, Tangshan, Jiujiang 1, Jiujiang 2 and Jiujiang 3. The DNA bands detected were analyzed by clustering analysis with SPSS software. RESULTS: The percentage of genetic similarity among the seven isolates was from 77.4% to 94.7%, showing a close genetic relationship among them. The percentages between the isolates of Beijing 1 and Tangshan, Jiujiang 1 and Jiujiang 2, Beijing 2 and Jiujiang 3 were 89.2%, 92.1% and 94.7% respectively, while that of Jiujiang 1 and Chengde was 77.4%, indicating a lower homology. CONCLUSION: There are a close genetic relationship and certain gene polymorphism among the seven T. vaginalis isolates; geographical origin plays little role to the genetic characteristics.

Animals↗

A RAPD-PCR genotyping assay which correlates with serotypes of group B streptococci.

AIMS: The purpose of this study was to determine if DNA polymorphisms generated by RAPD-PCR could be used to characterize Group B streptococci (GBS) for epidemiological purposes. METHODS AND RESULTS: 30 unrelated, previously serotyped strains were analysed by RAPD-PCR using two 10-mer primers (5' TGCGAGAGTC 3' and 5' AGAGGGCACA 3'). Both primers generated DNA electropherotype patterns which, on analysis, clustered the isolates within their respective serotypes. A blind test of a further 3 field isolates also defined these strains within their subsequently determined serotypes. The detection of DNA polymorphisms between isolates within a serotype confirmed previous reports of the heterogenous nature of individual GBS serotypes. CONCLUSIONS: The RAPD-PCR is a potentially useful assay for the rapid characterization of neonatal infections associated with group B streptococci. The method appears to be more discriminatory than conventional serological assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD-PCR assay is faster, more convenient and easier to perform than alternative DNA analytical procedures such as Pulsfield Gel Electrophoresis. We were able to reproduce the same results following re-testing of all isolates some 12 months later which suggests that the assay may be robust enough for use in routine epidemiological investigations.

Bacterial Typing Techniques↗

Variability in the pattern of random amplified polymorphic DNA.

The random amplified polymorphic DNA (RAPD) technique is a simple method to detect DNA polymorphism. It is sensitive to reaction conditions. Small changes in the reactants' concentration cause variations in amplification products. Using DNA from Asparagus officinalis, Dactylis glomerata, Mercurialis annua and Escherichia coli, we examined variability in the amplification pattern associated with reaction constituents. An increase in the ratio of Taq DNA polymerase to DNA in the reaction increased the number of amplified fragments. Increasing the concentration of primer resulted in the amplification of low molecular weight DNA fragments, while lowering the concentration resulted in high molecular weight fragments. Subsets of amplified fragments required different concentrations of magnesium for their highest intensity. Mechanical shearing of DNA obtained by sonication led to reduction in amplification of a subset of products. Enzymatic fragmentation of DNA by restriction enzymes led to loss or gain of specific fragments, depending on the DNA, primer, and restriction enzyme. RAPD markers of pooled DNA of anonymous pedigree should be critically evaluated for frequent 'false positive' markers.

Base Composition↗

Detection of genomic instability in lung cancer tissues by random amplified polymorphic DNA analysis.

Genomic instability resulting in multiple mutations is believed to be a driving force in the carcinogenic process. In this study, the random amplified polymorphic DNA (RAPD) technique, a simple PCR-based DNA polymorphism assay system, was used for detecting genomic instability in lung cancer tissues. DNAs from 20 lung cancer (18 non-small cell lung cancers and two small cell lung cancers) and their corresponding normal tissues were amplified individually by RAPD with seven different 10-base arbitrary primers. PCR products from RAPD were electrophoretically separated in agarose gels and banding profiles were visualized by ethidium bromide staining. The ability to detect genomic instability in 20 cancer tissues by each single primer ranged from 15 to 75%. DNA changes were detected by at least one primer in 19 (95%) cancer tissues. These results seem to indicate that genomic rearrangement is associated with lung carcinogenesis and that RAPD analysis is useful for the detection of genomic instability in lung cancer tissues.

Biopsy↗

Genotyping and antifungal susceptibility profile of Dipodascus capitatus isolates causing disseminated infection in seven hematological patients of a tertiary hospital.

Seven cases of disseminated infection due to Dipodascus capitatus are reported. Infections occurred in a hematological unit of a tertiary hospital during a period of 5 years. Five cases were refractory to antifungal therapy. Antifungal susceptibility testing of seven isolates was performed, and strains were typed by PCR fingerprinting with the core sequence of phage M13 and by random amplification of polymorphic DNA with two primers, Ap12h and W-80A. A very short range of MICs of each antifungal agent was observed. The MICs of amphotericin B ranged between 0.50 and 2 microg/ml. Strains were susceptible in vitro to flucytosine and susceptible (dose-dependent) to fluconazole and itraconazole. Voriconazole exhibited an activity in vitro comparable to that of itraconazole. Typing techniques allowed seven additional isolates of D. capitatus neither geographically nor temporally related to be classified into two different genomic patterns. The genomic type of the seven strains from the hematological unit was identical regardless of typing technique utilized. It would indicate that the seven cases of disseminated infection could be related epidemiologically.

Adult↗

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.

Animals↗