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PubMed1997

Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages.

A Burkholderia cepacia population naturally occurring in the rhizosphere of Zea mays was investigated in order to assess the degree of root association and microbial biodiversity at five stages of plant growth. The bacterial strains isolated on semiselective PCAT medium were mostly assigned to the species B. cepacia by an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (16S rDNA) (ARDRA) with the enzyme AluI. Partial 16S rDNA nucleotide sequences of some randomly chosen isolates confirmed the ARDRA results. Throughout the study, B. cepacia was strictly associated with maize roots, ranging from 0.6 to 3.6% of the total cultivable microflora. Biodiversity among 83 B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique with two 10-mer primers. An analysis of RAPD patterns by the analysis of molecular variance method revealed a high level of intraspecific genetic diversity in this B. cepacia population. Moreover, the genetic diversity was related to divergences among maize root samplings, with microbial genetic variability markedly higher in the first stages of plant growth; in other words, the biodiversity of this rhizosphere bacterial population decreased over time.

Base Sequence↗

PubMed1999

Antifungal susceptibilities and genetic relatedness of serial Trichophyton rubrum isolates from patients with onychomycosis of the toenail.

Onychomycosis is a common fungal disease infecting up to 20% of the population over age 40. The major causative agent of onychomycosis is Trichophyton rubrum. Uncontrolled infection may eventually lead to penetration of the newly forming nail plate. In spite of the encouraging cure rate with recent antifungal agents such as the allylamines (terbinafine) and azoles (itraconazole and fluconazole) some patients inevitably fail therapy. In this investigation, a group of patients from a multi-center study designed to assess the efficacy of terbinafine with known cases of onychomycosis were selected for evaluation. Nail samples from this patient group were colonized with T. rubrum throughout the terbinafine therapy. Antifungal susceptibility testing was performed on these T. rubrum isolates to detect change in MIC values. Strain relatedness was examined using random amplified polymorphic DNA (RAPD) technique. Our results revealed failure of patients to clear T. rubrum is not related to the development of resistance to the drug. While species determination was possible, we were not able to identify differences that would indicate reinfection with a new strain. Analysis of patient demographic data revealed that 70% of patients were over 45 years old, 56.6% were previously treated with antifungals, 60% came from family history with onychomycosis and 13% were diabetic. In conclusion, our data indicate that patients' failure to clear onychomycosis was not associated with resistant development. Failure of terbinafine therapy may be dependent on host-related factors.

Adult↗

PubMed1997

Typing of Candida albicans by use of a combined PCR/hybridization assay.

A modified genotyping assay for Candida albicans based on the RAPD-PCR (random amplification of polymorphic DNA) is described. Following capillary blot of RAPD-PCR products onto a nylon membrane, hybridization with a PCR-generated gene probe resulted in heterogeneous band patterns. By this method the discriminatory power of RAPD-PCR is enhanced with satisfactory reproducibility and reduced interference.

Candida albicans↗

PubMed2001

Phenotypic switching and genetic diversity of Cryptococcus neoformans.

Niger seed agar was used as a primary plating medium for the isolation of Cryptococcus neoformans from cerebrospinal fluid specimens from AIDS patients with untreated primary cryptococcosis. The medium was used as the primary means to detect variations in the colony morphology of the yeast. To search for phenotypic and genetic variations, nine patients individually harboring two or three types of colony morphology were studied. Intraindividual isolates from nine patients had minor variations in the API 20C profile, and the MICs of one or more antifungal agents (amphotericin B, fluconazole, and itraconazole) for isolates from three patients were significantly different. Intraindividual isolates from three patients had minor karyotype differences, and one showed a dramatic chromosomal length polymorphism. In addition, three serial isolates from a patient with two episodes of infection showed similar karyotypes, confirming persistent infection by the same strain. Random amplified polymorphic DNA products were identical for all isolates (including three isolates from a relapse case). Our results provided evidence suggesting that (i) in humans, C. neoformans may undergo phenotypic and genetic changes during early infection prior to antifungal agent administration; (ii) dramatic variations in electrophoretic karyotypes and in phenotypes, as demonstrated during the early infection of one patient, may be due to infection by different strains; and (iii) the use of niger seed agar as a primary plating medium is useful for studying antifungal susceptibility, phenotypic switching, genetic diversity, and multiple-strain infections.

AIDS-Related Opportunistic Infections↗

PubMed1997

Molecular typing and fluconazole susceptibility of urinary Candida glabrata isolates from hospitalized patients.

At our community teaching hospital between August 1994 and August 1995, Candida glabrata accounted for 14% of all Candida isolates and for 31% of urinary Candida isolates. The culture site was urine for 68% of C. glabrata isolates compared to 30% of all Candida isolates (p < 0.001, chi 2). To study the association between C. glabrata and isolation from the urine, we analyzed all available C. glabrata urinary isolates over a 3-month period (23 isolates from 20 patients) using electrophoretic karyotyping, random amplified polymorphic DNA analysis, and fluconazole susceptibility testing. Random amplified polymorphic DNA generated eight types, although electrophoretic karyotyping generated 17 types. Combining the two methods resulted in 19 types indicating that urinary C. glabrata strains at our hospital are genetically diverse and the association between C. glabrata and urinary tract isolation does not appear to be due to horizontal transmission of a single or small number of strains. In vitro susceptibility tests showed that C. glabrata isolates from patients receiving fluconazole had significantly higher minimum inhibitory concentrations to fluconazole than those not receiving fluconazole (p < 0.05). Despite a limited number of patients and isolates, our data suggest that selection of less susceptible organisms by the presence of antifungal agents may be an important contributor to increased urinary isolation of C. glabrata from patients in our hospital.

Antifungal Agents↗

PubMed2001

Use of molecular markers to study the effects of environmental impacts on genetic diversity in brown bullhead (Ameirus nebulosus) populations.

Conservation biology needs sound biological information in order to maintain biological diversity in the face of the current rate of loss. An important component of the information needed is the level of genetic diversity within and between populations, especially for those species faced with exposure to environmental stressors. We applied multilocus DNA profile analysis (highly variable number tandem repeats [HVNTR] and randomly amplified polymorphic DNA [RAPD] techniques) and allozyme analysis to test whether individuals from historically degraded sites display levels of genetic diversity different from individuals taken from reference sites. Four Lake Erie tributaries, two impacted and two reference sites, were the sources of brown bullhead (Ameiurus nebulosus) samples. Pairwise comparison of the sampled populations demonstrated an association of decreased genetic diversity with exposure of brown bullhead to stressors using both RAPD and HVNTR analysis.

Animals↗

PubMedDate not supplied

Inheritance pattern of RAPD markers in Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae).

Melipona quadrifasciata is an important pollinator agent in several regions of Brazil. Data concerning the genetics of this species are scarce in the literature. In this work we used the random amplified polymorphic DNA (RAPD) technique to determine the degree of polymorphism and the inheritance pattern of these molecular markers in this species. Our ultimate goal is to establish tools to be used in the study of the genomic organization of M. quadrifasciata. Genomic DNA from progenies F(1) and BC(1) were assayed with 79 different primers, yielding an average of 6.67 bands and 1.68 polymorphisms per primer. Three types of polymorphisms were detected: band presence/absence, band intensity, and fragment-length polymorphisms. Most of the observed polymorphisms were band presence/absence, typical of RAPD-dominant markers. The number of observed polymorphisms and their segregation in accordance with a Mendelian proportion confirm the importance of this technique for genome analysis of species like M. quadrifasciata that are poorly studied at the genetic level.

Animals↗

PubMed1998

Pseudomonas aeruginosa from cystic fibrosis patients: study using whole cell RAPD and antibiotic susceptibility.

Pseudomonas aeruginosa is the most important bacterial pathogen in lung disease of cystic fibrosis patients. Different morphotypes of the bacterium are frequently recovered in sputum samples of these patients. We developed a whole cell Randomly Amplified Polymorphic DNA (RAPD) technique in order to establish the relatedness between morphotype, genotype and antibiotic susceptibility. Six cystic fibrosis patients already colonized by P. aeruginosa were investigated by collecting three successive sputum samples (before and after antibiotic treatment, and one month later) and selecting 10 isolates per morphotype. 250 isolates of P. aeruginosa were recovered from 16 of 18 sputum samples. Five patients carried a single RAPD type strain four of which showed at least two morphotypes; one patient carried two RAPD types strains. No patients carried the same strain. These results confirmed other studies previously published in showing stability of the chronic colonization with a single strain. Antibiotype differences were not associated with differences of RAPD profiles and no relation was found between antibiotype and morphotype.

Cystic Fibrosis↗

PubMed2001

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE).

Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.

Animals↗

PubMed2002

Random amplified polymorphic DNA analysis for differentiation of Leuconostoc mesenteroides subspecies isolated from Tenerife cheese.

AIMS: In the present study, a RAPD-PCR fingerprinting method was developed to assign Tenerife cheese Leuconostoc mesenteroides strains to its three subspecies (mesenteroides, cremoris and dextranicum). METHODS AND RESULTS: Arbitrarily primed-PCR gave different DNA banding patterns for each type strain of Leuc. mesenteroides subspecies consisting in three major and intense bands of: 1800, 1600 and 1150 bp for subspecies mesenteroides 1800, 1150 and approximately 350 bp for subspecies cremoris; and 1800, 1600 and 1500 bp for subspecies dextranicum. DNA fingerprints of Tenerife cheese Leuc. mesenteroides subspecies were coincident to that of their respective type strain. RAPD profiles were reproducible with DNA template obtained by two different extraction methods. CONCLUSIONS: Tenerife cheese Leuc. mesenteroides strains were rapidly and unequivocally assigned to one of the subspecies by comparing their RAPD-PCR fingerprints with those displayed by type strains used as standards. This technique can be applied to complement preliminary identification of Leuc. mesenteroides to the species level by other molecular methods such as protein fingerprinting. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD-PCR allows reliable, reproducible and rapid molecular differentiation of Tenerife cheese Leuc. mesenteroides subspecies with no need to use time-consuming and often ambiguous biochemical tests.

Bacterial Typing Techniques↗

PubMed1998

Strain-specific identification of probiotic Lactobacillus rhamnosus with randomly amplified polymorphic DNA-derived PCR primers.

In the present work, strain-specific PCR primers for Lactobacillus rhamnosus Lc 1/3 are described. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. They were screened for specificity by hybridization with DNA from 11 L. rhamnosus strains. A 613-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific to L. rhamnosus Lc 1/3 was constructed based on the sequence. The primer pair was tested with 11 Lactobacillus species and 11 L. rhamnosus strains and was found to be strain specific. The nucleotide sequence of the specific RAPD marker was found to contain part of a protein encoding region which showed significant similarity to several transposases for insertion sequence elements of various bacteria, including other lactic acid bacterium species.

Amino Acid Sequence↗

PubMed2006

[Cloning and analyzing of the female-specific marker in the dioecious species Asparagus officinalis L].

Sex-linked molecular markers are being obtained, which would be essential to be used in the screening of different sex of dioecious plants at the seedling stage. Furthermore, it is important in cloning the gene related to the sex. In this study the random amplified polymorphic DNA (RAPD) technique was employed with the objective to find markers linked to sex determination in Asparagus. A total of 100 primers were tested with the same PCR cycling procedure. A female-associated fragment with a length of about 867bp was generated with S12 primer. The fragment was cloned and sequenced, showing it is abundant in AT and contains 2 shorter open reading frames. In order to convert the RAPD marker into SCAR (sequence characterized amplified regions) marker, 24bp specific primers were constructed and used for PCR amplifying. The female-linked dominant SCAR marker was obtained, which would be efficient to identify the different sex of Asparagus officinalis L.

Amino Acid Sequence↗

PubMed2004

Molecular characterization of contaminant-indicative RAPD markers.

This paper describes genetic markers which can be used to study selection and genetic adaptation of organisms to radionuclide and other types of contaminant stress. Previous research using the randomly amplified polymorphic DNA (RAPD) technique has identified several markers which revealed genetic differences between contaminated and reference western mosquitofish (Gambusia affinis) populations. Experimental evidence suggested that these markers may be associated with loci involved in determining relative fitness in radionuclide-contaminated environments ("contaminant-indicative markers"). In the present study, Southern blot analyses show these markers to be highly conserved in DNA sequence and molecular length in sea urchins, mosquitofish, herring gulls and humans. Such conservation is thought to be rare among RAPD bands. Results of DNA sequencing efforts did not provide definitive evidence as to the identity of these loci, but indicated that short segments (<40 bp) of known DNA sequences were homologous to various regions of the RAPD sequences. Furthermore, the regions of homology seemed to be non-randomly distributed along the length of the RAPD markers. Although the identity of these bands is still unknown, the high degree of conservatism suggests that these loci might play an important role in molecular processes.

Animals↗

PubMed2004

Phylogenetic relationships among genotypes of worldwide collection of spring and winter ryes (Secale cereale L.) determined by RAPD-PCR markers.

Genetic similarities among 20 spring and 22 winter accessions of agronomically different ryes from fourteen countries were estimated by employing random amplified polymorphic DNA (RAPD) techniques. Cluster analysis of genetic distance data showed that 42 genotypes were readily classifiable into two main groups: spring and winter groups. Within the spring group, cultivars fell into a North European and an American-Chinese group. Cultivars of winter rye fell into four groups: Northern European, Russian, American and Chinese lines. A UPGMA-dendrogram based on genetic distances of cultivars of rye within the winter and spring groups showed that the clusters corresponded well to their geographical locations. The results indicated that isolation has played an important role in the evolution of rye, and that temporal isolation has influenced the genetic diversity of rye more than geographical isolation. In this experiment, RAPD proved to be a rapid, reliable and practicable method of revealing polymorphisms in rye populations.

Ecosystem↗

PubMed2000

Genetic variation within and among patches of the clonal species, Vaccinium stamineum L.

Once thought to be dominated by a few genets, clonal plant populations can contain high levels of genetic diversity. Sexual reproduction and vegetative growth strategy affect the amount and distribution of genetic diversity within clonal plant populations. We determined the scale of genetic diversity in a population of Vaccinium stamineum, a clonal shrub that forms discrete patches. Using the random amplified polymorphic DNA (RAPD) technique, we surveyed the genetic diversity of V. stamineum within and among patches from a 1-ha site. We found 67 unique RAPD profiles among the 99 sampled individuals from 22 patches. In two patches, all the sampled individuals had the same RAPD profile. In seven patches, every individual sampled had a different RAPD profile. The remaining patches showed mixed RAPD profiles which suggested both clonal and sexual reproduction. Each unique RAPD profile was restricted to one patch (with one exception), which suggests that clonal growth occurs at the patch scale. High levels of genetic variation within some patches may be explained by somatic mutation; however, seedling recruitment is a more likely explanation.

DNA, Plant↗

PubMed1998

Application of random amplified polymorphic DNA PCR for genomic analysis of HIV-1-infected individuals.

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) is a DNA fingerprinting technique used to detect genomic polymorphisms. We employed sixteen different RAPD-PCR 10-mer primers to amplify DNA from the peripheral blood mononuclear cells (PBMC) of 80 HIV-1-infected individuals. These individuals were previously identified as either heterozygotes (+ /delta32) and homozygotes (+/+) for the CCR5 locus by PCR with gene specific primers. Four of the sixteen randomly selected RAPD primers produced distinguishable banding profiles between CCR5 (+/delta32) heterozygotes and CCR5 (+/+ ) homozygotes. Direct sequencing of some RAPD-PCR products obtained with one of the four RAPD primers that were tested yielded clearly readable, but limited sequences, which were similar to portions of the previously published sequences for (+/+ ) homozygotes (98% similarity) and (+/delta32) heterozygotes (87% similarity) of the CCR5 alleles. Thus, the RAPD-PCR technique may be useful for the identification of human molecular markers that may correlate with susceptibility to HIV-1-infection, or differences in disease progression among HIV-l-infected individuals.

Base Sequence↗

PubMed2006

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria.

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.

Bifidobacterium↗

PubMed1999

Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis.

The random amplified polymorphic DNA (RAPD) technique was employed in the haplo-diploid dioecious species Gracilaria gracilis to identify sex-linked PCR markers. Sixty-nine decamer oligonucleotide primers were tested on two bulks of DNA, one from five haploid males and the other from five haploid females. One of these primers (OPD13) generated a 430-bp fragment specific to males and a 620-bp fragment specific to females. The diploid individuals (tetrasporophytes) showed the co-occurrence of these two fragments. In order to verify the linkage between the sexual phenotypes and these markers, a progeny array of 59 haploid individuals (male and female) born on a diploid individual was analysed, in all of which the two markers produced by the OPD13 primer segregated perfectly with sex.

Base Sequence↗