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At least 19 recordsLinked to original sources
PubMed1994

Characterization of Bacillus licheniformis with the RAPD technique (randomly amplified polymorphic DNA).

RAPD technique (randomly amplified polymorphic DNA) was used for epidemiological subtyping of Bacillus licheniformis and other Bacillus spp. Within 46 isolates of B. licheniformis, up to 10 strain types could be determined when two 10-mer primers were used. RAPD patterns, which were found in eight further strains of Bacillus spp., clearly differed from those of B. licheniformis. Thus RAPD technique proved to be a promising tool for characterization of Bacillus spp.

Bacillus↗
PubMed1993

Random amplified polymorphic DNA technique for the identification of Trichinella species.

The random amplified polymorphic DNA (RAPD) technique was successfully used to produce genetic fingerprints distinguishing between Trichinella spiralis and Trichinella britovi. The same patterns were obtained from purified and crude DNA preparations of pooled and single muscle larvae. RAPD fingerprinting was applied to muscle larvae preserved under different conditions and recovered from different hosts. Larvae recovered from fresh and frozen meat and stored at -20 degrees C for a long time or under 70% ethyl alcohol at room temperature for 30 d gave good and reproducible results. Single larvae recovered from a naturally infected wild boar and from a human biopsy gave fingerprints congruent to those obtained from T. britovi reference strains. The results prove that RAPD analysis is a quick method to distinguish between the autochthonous Trichinella species of Central-Southern Europe in less than 1 d after the detection of the infection. If necessary, the biological material can be frozen or stored under 70% ethyl alcohol at room temperature and sent to laboratories able to perform the RAPD analysis. The RAPD technique requires no prior knowledge of the molecular biology of the organism to be investigated and therefore appears to be a promising tool in parasitology for the identification of sibling species.

Animals↗
PubMed1996

Analysis of Heterodera avenae populations by the random amplified polymorphic DNA technique.

Eleven populations of the Heterodera avenae complex (four Spanish, two British, two French, and three Swedish) were studied by random amplified polymorphic DNA analysis. From 5 to 11 fragments were obtained with each of 14 random primers, with fragment size ranging from 200 to 2200 bp. Cluster analysis of the 11 populations, using 108 scorable markers, separate these populations into two main groups. These groups coincide with what is known as the "true" H. avenae and the "Gotland strain" or "British pathotype 3" of H. avenae. The results also clarify the relationships among some members of the H. avenae complex established previously using morphological and biochemical criteria.

Animals↗
PubMed1994

Comparative genetic analysis of Swiss and Spanish isolates of Echinococcus granulosus by southern hybridization and Random Amplified Polymorphic DNA technique.

Swiss and Spanish isolates of Echinococcus granulosus were compared using different molecular biological techniques: Genomic DNAs isolated from parasites originating from various intermediate hosts were subjected to Southern hybridization with different probes, the same source of DNA was used for DNA amplification using the Random Amplified Polymorphic DNA (RAPD) technique. With both methods the various isolates (metacestodes) of E. granulosus exhibited characteristic banding patterns which allowed us to assign them to the following groups of homologous profiles: (a) isolates of horse and donkey origin from Spain and Switzerland; (b) isolates of cattle origin from Switzerland; (c) isolates of sheep, cattle and human origin from Spain; (d) isolates of pig origin from Spain and Switzerland and of goat origin from Spain. By RAPD (Southern hybridization not examined) two isolates of human origin from Switzerland were showing banding patterns distinct from groups (a-d). The results provide further evidence that the morphological and biological differences of several strains of E. granulosus are also detectable on the genetic level using molecular biological techniques.

Animals↗
PubMed1993

Application of the random amplified polymorphic DNA technique for the detection of polymorphism among wild and cultivated tetraploid wheats.

Development of a high-density genetic linkage map of cultivated wheats using conventional molecular markers has lagged behind the other major food crops such as rice and tomato because of the large genome size and limited levels of genetic polymorphisms. Recently, random amplified polymorphic DNAs (RAPDs) have been suggested to provide an alternative to visualize more polymorphism. For the construction of a genetic linkage map in tetraploid wheats, one can use a strategy of intersubspecific crosses between the most dissimilar wild and cultivated tetraploid wheats that are easy to hybridize and result in fertile progeny. An assessment of the level of RAPDs among different accessions and varieties of wild and cultivated tetraploid wheats is required to fulfill this objective. We present here the data obtained using RAPD analysis of 40 primers in 20 accessions of wild tetraploid emmer wheats (Triticum turgidum L. ssp. dicoccoides) and 10 genotypes of cultivated tetraploid durum wheats (Triticum turgidum L. ssp. durum) selected from geographically diverse locations. We have observed a higher level of polymorphism among different accessions of wild emmer wheat from Israel. Turkey, and Jordan than the group of cultivated American, Turkish, and Syrian durum wheats. These data have been used to generate a dendrogram suggesting the genetic relationships among these genotypes, and the most dissimilar genotypes are identified for future mapping and gene tagging work.

Base Sequence↗
PubMed1996

Use of random amplified polymorphic DNA (RAPD) technique in inheritance studies of Plasmodium falciparum.

Effectiveness of random amplified polymorphic DNA (RAPD), a technique using 1 10-base primer to amplify random segments of genomic DNA, and some of its possible uses were tested in the A + T-rich genome of Plasmodium falciparum. The best concentrations of MgCl2, 60% G + C primer, and DNA were determined to be 4.0 mM, 0.4 microM, and 90-180 ng/15 microliters reaction, respectively. Use of 30% G + C primers did not allow amplification to occur. Application of RAPD to DNA of parent and progeny clones from a P. falciparum cross showed that polymorphisms identified in the parentals and tracked in the progeny were inherited in a Mendelian fashion and that RAPD-identified polymorphisms could be used as genetic markers. Some of these polymorphic markers were located on more than 1 chromosome, whereas others were specific for a single chromosome. Two of these markers, each located on chromosome 3 of 1 of the parental parasites, were missing from 2 of the 18 progeny, suggesting that deletions, or crossover events had occurred. RAPD markers also identified a higher number of nonparental-type progeny than expected, thus confirming previous observations for high genetic variability in malaria parasites.

Animals↗
PubMed1995

Genetic characterization of Toxoplasma gondii strains by random amplified polymorphic DNA polymerase chain reaction.

The technique of Random Amplified Polymorphic DNA (RAPD) PCR has been used to detect DNA polymorphisms among Toxoplasma gondii strains. Seven arbitrary oligonucleotides (10-mer) were used as primers to amplify total genomic DNAs and significant genetic heterogeneity was detected among 11 T. gondii strains with different virulence for mice. The polymorphisms observed allowed relationship dendrograms of T. gondii strains to be constructed by PHYLIP and PAUP analyses. The genetic relationships of the T. gondii strains generated by 2 analyses using completely different assumptions were similar. Both analyses revealed 2 groups of T. gondii strains, one formed by the 6 virulent strains and the other formed by the 5 avirulent strains. This suggests that the genus Toxoplasma may actually contain 2 groups, correlated with their virulence, which have probably evolved independently following their initial separation. Significant polymorphisms were also detected between 2 different laboratory stocks of the T. gondii RH strain.

Animals↗
PubMed1996

A total DNA characterization in Proteocephalus exiguus and P. percae (Cestoda: Proteocephalidae): random amplified polymorphic DNA and hybridization techniques.

Two morphologically similar fish tapeworms, Proteocephalus exiguus and P. percae, were differentiated by the random amplified polymorphic DNA (RAPD) marker and Southern-blot hybridization techniques. Four geographic isolates of P. exiguus and two of P. percae were studied using eight arbitrary decamer oligonucleotides as primers. Species-specific RAPD fragments enabled reliable separation of the taxa studied. Generally, 1-3 fragments specific for P. percae and 1-4 P. exiguus-specific fragments were detected using individual primers. Intraspecific differences in P. percae depended on the primer used. The profiles generated by the primers OPA 04 and OPA 12 showed an intraspecific variation, whereas six other primers tested revealed indistinguishable banding patterns. On the other hand, intraspecific variability of P. exiguus, detected among four geographic isolates, was evident using all primers. Additionally, the utilization of two species-specific RAPD markers as effective probes was demonstrated. Distinct differences between P. exiguus and P. percae were revealed using each of the restriction enzyme/probe combinations.

Animals↗
PubMed1995

Phenotypic and genotypic analysis of variability in Aspergillus fumigatus.

Sixty-one isolates and collection strains of Aspergillus fumigatus were compared for their phenotypic (morphological features and isoenzyme profiles) and genotypic (restriction enzyme-generated mitochondrial DNA and ribosomal DNA profiles and random amplified polymorphic DNA patterns) features. The examined strains exhibited highly variable colony morphologies and growth rates at different temperatures, but their micromorphologies and conidial diameters were characteristic of the species. Of the isoenzymes studied, the beta-arylesterase and phosphatase patterns were the most divergent, and the 61 strains could be classified into seven groups. The glucose 6-phosphate dehydrogenase and catalase isoenzyme patterns displayed only a limited variability, while the profiles of superoxide dismutase, lactate dehydrogenase, and glutamate dehydrogenase were highly conserved. The HaeIII-generated mitochondrial DNA patterns and SmaI-digested repetitive DNA and ribosomal DNA hybridization patterns of almost all strains were also invariable. The level of variation was much higher when random amplified polymorphic DNA analysis was applied. Although the patterns of the strains were very similar with most of the primers, the application of some primers made it possible to cluster the A. fumigatus isolates into several groups. The results indicate that the random amplified polymorphic DNA technique could be used more efficiently than isoenzyme analysis for typing A. fumigatus isolates. A good correlation was found between the dendrograms obtained from the isoenzyme and random amplified polymorphic DNA data, but the isoenzyme and amplified DNA patterns did not correlate with the pathogenicity, pigment production, or geographical origin of the strains. One "A. fumigatus" strain (strain FRR 1266) exhibited unique isoenzyme, mitochondrial DNA, ribosomal DNA, and random amplified polymorphic DNA patterns; it is proposed that this strain represents a new species of the section Fumigati.

Aspergillus fumigatus↗
PubMed1995

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii.

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.

Base Sequence↗
PubMed1996

Species, sub-species and hybrid populations of the blowflies Lucilia cuprina and Lucilia sericata (Diptera:Calliphoridae).

The blowflies Lucilia cuprina Wiedmann and Lucilia sericata Meigen (Diptera:Calliphoridae) are facultative ectoparasites of warm blooded vertebrates, particularly domestic sheep. Despite being similar in morphology and ecology, the two species and different populations of each species, are known to vary in their importance as pests in different regions of the world. To elucidate the genetic basis of these species and population level differences, flies were collected from sites in Africa, Europe, Australasia, North America and the islands of Hawaii, and examined using a combination of morphology, the random amplified polymorphic DNA technique and a complementary mitochondrial DNA analysis. The results confirm the species integrity of L. sericata and L. cuprina and support the existence of intra-specific genetic variation in L. cuprina, but not L. sericata.

Animals↗
PubMed1996

Nosocomial CDC group IV c-2 bacteremia: epidemiological investigation by randomly amplified polymorphic DNA analysis.

The CDC group IV c-2 bacterium is a gram-negative bacillus rarely isolated from clinical specimens. This organism caused catheter-related bacteremia in five immunocompromised children hospitalized in two distinct wards of our institution between November 1993 and October 1994. Three patients recovered on empiric antibacterial chemotherapy combining ceftazidime and amikacin, and a fourth patient required imipenem instead of ceftazidime. The fifth patient recovered without treatment. Catheter removal was never necessary. The randomly amplified polymorphic DNA technique with three different primers was applied to nine isolates recovered by culturing blood from the five children and showed that all of the patients harbored isolates of the same genotype. The source of the outbreak could not be determined.

Adolescent↗
PubMed1996

["Arbitrarily primed PCR". Or: chance in the service of science].

"Arbitrarily primed PCR" (AP-PCR) methods are based on the amplification of DNA with arbitrarily selected primers. In the present review article two selected applications of this methodical approach are summarized. Using the RAPD ("Random Amplified Polymorphic DNA") technique the genetic variability of different organisms can be analyzed by generating a genomic fingerprint. Echinococcus granulosus isolates (metacestodes) from Spain and Switzerland were comparatively characterized by the use of this molecular epidemiological tool. The following groups of genetically related isolates could be identified: (a) equine isolates (horse/donkey) from Spain and Switzerland, (b) cattle isolates from Switzerland, (c) goat isolates from Spain and pig isolates from both countries, (d) sheep, cattle and human isolates from Spain. Isolates derived from Swiss patients were forming a separate group with respect to their genetic relatedness. As a further application of the AP-PCR, the development of a novel technique for the construction of cDNA libraries from minute amounts of starting material using an Echinococcus multilocularis metacestode library as an example is presented.

Animals↗
PubMed1995

The combination of Gilbert/Maxam chemical sequencing and the dideoxynucleotide chain termination approach facilitates the construction of species specific PCR-primers based on diagnostic RAPD bands.

The randomly amplified polymorphic DNA technique (RAPD) is a modification of PCR that uses short, arbitrarily generated single primers to amplify genomic DNA. Amplified DNA-fragments are often polymorphic and can be used as individual, population- or species-specific markers. Because the RAPD technique requires a very high degree of reproducibility at the instrumentation level and with regard to buffer conditions, we propose to synthesize highly specific conventional PCR primers, the sequence of which is based on the primary diagnostic RAPD bands. In this communication we present a fast and convenient experimental strategy for converting the non-stringent RAPD conditions with their low annealing temperatures to stringent PCR conditions. Diagnostic RAPD bands were sequenced by a combination of chemical (Gilbert/Maxam) and chain termination (Sanger) techniques. Based on this sequence information, highly specific oligonucleotide primers were synthesized. The value of this approach was demonstrated for the molecular diagnosis of the important rape seed (Brassica napus) pathogen Leptosphaeria maculans.

Ascomycota↗
PubMed1996

Echinococcus granulosus: genomic and isoenzymatic study of Spanish strains isolated from different intermediate hosts.

The phenomenon on intraspecific variation in Echinococcus granulosus is already documented in Spain, where unilocular hydatidosis is an endemic disease. The first speciation studies, focused at a genomic level, showed the existence of three different strains: ovine-bovine-human, equine and swine-caprine. In the present study, the genomic identification, by random amplified polymorphic DNA technique (RAPD) of a larger number of Spanish E. granulosus isolates, using five different primers, showed the maintenance of these groups. Thus, some of these strains may not be infective for man. These conclusions were supported by a phenotypic characterization of the same isolates by zymodeme technique, showing the five isoenzyme systems used that Spanish E. granulosus strains can also be distinguished at a phenotypic level by isoenzymatic patterns. Both techniques (RAPD and zymodemes) were used for statistical analysis and for the construction of two dendrograms, which were slightly different. In addition, some intrastrain variation was detected with both techniques, a phenomenon that is directly related to the different speciation theories proposed for E. granulosus strains. The epidemiological implications of the results are discussed in the text.

Animals↗
PubMed1996

Last male sperm precedence in a damselfly demonstrated by RAPD profiling.

We used the random amplified polymorphic DNA technique to determine last male sperm precedence (P2) in the damselfly Calopteryx splendens xanthostoma (Charpentier). We amplified DNA from mothers, putative fathers and from the embryos of individual offspring, and subsequently calculated band-matching coefficients between known first-order relatives (offspring within a clutch) and non-relatives (mothers and fathers) to estimate last-male paternity. The data indicate that, as in other Calopterygidae, P2 is high (0.98) in the bout of oviposition immediately following copulation, despite the fact that the males of this species do not completely remove the sperm of previous males (Siva-Jothy & Hooper 1995).

Animals↗
PubMed1996

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA.

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.

Animals↗