Hourly activity of Lutzomyia ovallesi and L. gomezi (Diptera:Psychodidae), Vectors of cutaneous leishmaniasis in northcentral Venezuela

Random amplified polymorphic DNA (RAPD) analysis was used to amplify the genome of black tiger prawns (Penaeus monodon) to detect DNA markers and assess the utility of the RAPD method for investigating genetic variation in wild P. monodon in Thailand. A total of 200 ten-base primers were screened, and 84 primers yielded amplification products. Six positive primers that gave highly reproducible RAPD patterns were selected for the analysis of three geographically different samples of Thai P. monodon. A total of 70 reproducible RAPD fragments ranging in size from 200 to 2000 bp were scored, and 40 fragments (57%) were polymorphic. The RAPD analysis of broodstocks from three different locales, Satun-Trang, Trat, and Angsila, revealed different levels of genetic variability among the samples. The percentages of polymorphic bands were 48% and 45% in Satun-Trang and Trat, respectively, suggesting a high genetic variability of the two samples to be used in selective breeding programs. Only 25% polymorphic bands were found in the Angsila sample, indicating the lowest polymorphic level among the three samples examined. Primer 428 detected a RAPD marker that was found only in P. monodon originating from Satun-Trang, suggesting the potential use of this marker as a population-specific marker in this species.     

RAPD typing of Campylobacter jejuni and comparison with Lior's or Penner's serotyping system

Campylobacter jejuni were isolated from 7 epidemic outbreaks (121 isolates), 15 patients with gastroenteritis, chicken meats (47 isolates) and chicken cecal contents (70 isolates). The isolates and one standard strain of C. jejuni JCM2013 were analysed by randomly amplified polymorphic DNA method (RAPD). Total of 254 C. jejuni isolates were divided 68 different RAPD types which included strains that did not to divided by Lior's or Penner's serotyping system. To compare the similarities of RAPD patterns among the isolates, the amplification patterns of DNA were estimated by means of the Dice coefficient, and clustering of strains was based on the unweighted average pair group method (UPGMA) to facilitate the plotting of a dendrogram. It suggests that amplification band patterns of human isolates were different from those of chicken ones. Thus additional information given from RAPD profiles serves for epidemiological investigation and RAPD analysis is recommended as rapid and effective typing method.     

A fatal case of alimemazine poisoning

The genetic diversity of two samples of Cestoda (Bothriocephalus funiculus, Renaud and Gabrion, 1984) parasitizing two sympatric teleostean species was assessed using random amplified polymorphic DNA (RAPD). A total of 72 Bothriocephalus were analyzed individually, and electrophoretic analysis of the amplification products of 65 primers among the 68 tested revealed monomorphic patterns, reflecting the close genetic relatedness within and between the parasites of the two samples. However, 3 primers showed polymorphic patterns at 6 RAPD sites. Analysis of the distribution of these genomic fragments, assuming random mating, showed strong linkage disequilibria (only 8 genetic combinations were observed among the 32 expected). Two genetic entities displaying a high degree of host specificity were evidenced within our two samples of funiculus. This powerful molecular technique can be used as a diagnostic tool in studies concerning the biodiversity of related genetic entities and could have broad applications in parasitology.     

Authentication of the Chinese drug "ku-di-dan" (herba elephantopi) and its substitutes using random-primed polymerase chain reaction (PCR)

DNA fingerprinting and polymorphism among the Chinese drug "Ku-Di-Dan" and its substitutes were demonstrated with arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA (RAPD). Distinctive, reproducible genomic fingerprints from DNA of the Chinese drug Ku-Di-Dan and 9 species of Compositae were generated with six long (18-24 mer) and one short (10 mer) random-chosen primers with PCR. Ku-Di-Dan samples can be distinguished according to the banding patterns of their amplified DNA on agarose gels. Results showed that AP-PCR and RAPD fingerprints of the commercial samples of Ku-Di-Dan retailed in Fujian, Taiwan, Hong Kong and Macau markets are identical with that of Elephantopus scaber L. The relatedness of Ku-Di-Dan among 9 plants may also be estimated by the Similarity Indexes values of the genomic DNA fingerprints.     

A randomized, double-blind, placebo-controlled, crossover study to assess the immediate effect of sublingual glyceryl trinitrate on the ankle brachial pressure index, claudication, and maximum walking distance of patients with intermittent claudication

The conventional method of identifying acaricide resistance in a suspect tick population by the United Nations Food and Agriculture Organization packet assay is a laborious and time-consuming process. DNA probes have been demonstrated as rapid and accurate tools for detecting pesticide resistance in insect species. Random-amplified polymorphic DNA (RAPD) has been used by other groups to differentiate species of mosquitoes and populations within a mosquito species. By using different arbitrary oligonucleotides as primers with RAPD, we have demonstrated that various strains of Boophilus microplus (Canestrini) show different patterns of DNA fragments on agarose gel electrophoresis. The unique DNA fragments may be useful for developing probes that can detect acaricide resistance in field pest populations.     

Nucleotide sequence and genomic organization of repeating units of the AT-rich family or repeats in the diploid wheat Triticum monococcum L

In diploid wheat Triticum monococcum L., a novel family of AT-rich repeated DNA sequences arranged in clusters of tandem arrays of 560-bp fragments is described. The nucleotide sequence of a 556-bp repeat unit was determined. The basic structural unit of this repeated DNA family contains subrepeats with the main motif AAACATTGTTAC. This type of repetitive DNA sequence could evolve via tandem amplification of short subrepeats with some subsequent divergence, and/or via recombination with unique sequences. Structural organization of homologous sequences, revealed by blot hybridization in other representatives of the Triticeae tribe, suggest that this family of repeated DNA is genome-specific.     

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.     

Species differentiation by internally transcribed spacer PCR and HhaI digestion of fluconazole-resistant Candida krusei, Candida inconspicua, and Candida norvegensis strains

PCR amplification of the regions containing the internally transcribed spacers and 5.8S rRNA gene of Candida krusei, C. inconspicua, and C. norvegensis yielded fragments of 510, 460, and 500 bp, respectively. HhaI digestion of these fragments yielded species-specific bands. Random amplification of polymorphic DNA with primer R108 showed interspecific discriminatory band patterns. Susceptibilities to fluconazole and amphotericin B were determined.     

(-)Deprenyl (selegiline), a catecholaminergic activity enhancer (CAE) substance acting in the brain

Fast and reliable identification of different species of the genus Candida is important to define adequate therapeutic decisions, because the different species have highly variable susceptibilities to antifungal drugs; azoles and amphothericin B. Accurate statistical records on case history and epidemiological studies also depend on effective identification. To address this problem we established a RAPD method that enabled direct identification of five very common species of Candida. Initially, reference band patterns were established for C. albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. One of the primers, M2, showed remarkably conserved intra-specific patterns of approximately 10 bands each, ranging in size from 2.0 to 0.1 kb. These patterns were significantly different and species-specific. Few bands were conserved between different species of Candida, which was assumed to be consistent with their phylogenetic relatedness. In addition, band patterns were constant and reproducible and DNA isolated from single colonies yielded sufficient DNA for identification. The reference band patterns were then used, in blind experiments, to identify species of Candida in 50 randomly chosen samples, including clinical isolates and ATCC strains. RAPD results were 100% consistent with results obtained by conventional diagnostic methods and were achieved in one day instead of several days taken by conventional methods. Because ideal identification methods should be consistent with phylogeny and taxonomy we tested whether RAPD could be used to calculate genetic distances. Comparison of RAPD phylogenetic trees with 18S rRNA trees showed significant differences in tree topologies which indicated that RAPD data could not accurately measure the relative distances between different species. Also, computer simulations of RAPD random patterns were used to test whether the observed degree of RAPD band pattern similarities could occur at random. These simulations suggested that the level of inter-specific band pattern similarities observed in our data could be obtained at random, while intraspecific pattern similarities could not. RAPD would be helpful to discriminate between isolates but not to quantitate the differences. We suggest that the inaccurate estimate of genetic distances from RAPD is a general limitation of the technique and not a specific problem of our identification method. Because of the repetitive character of the target sequences, genetic distances calculated from RAPD could be affected by paralogy, namely, recombination and duplication events not parallel with speciation events.     

Characterization of clinical isolates of Streptococcus agalactiae by random amplified polymorphic DNA using degenerate oligonucleotides

Epidemiological studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemiologically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemiologically-unrelated isolates (classified into 5 different serotypes) resulted in 11 distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.     

Is the coccoid form of Helicobacter pylori viable?

Helicobacter pylori was grown on solid medium for up to 5 weeks. Morphological conversion from spiral to coccoid forms began after 7 days incubation under microaerophilic conditions. Similar to the exponential cultures, the ageing bacteria produced alkaline phosphatase, acid phosphatase, leucine arylamidase and naphthol-AS-beta 1-phosphohydrolase, although there was a reduction in the levels of the latter two enzymes. Unlike the other enzymes, urease was not detected in 5-week-old cultures. By using primers based on urease subunit C and 26 kD protein genes for polymerase chain reaction (PCR) these two important gene fragments remained conserved despite the morphological conversion from spiral to coccoid forms. Furthermore, PCR-based random amplified polymorphic DNA (RAPD) fingerprinting showed similar DNA banding patterns from bacteria of various ages, demonstrating the conservation of the DNA composition despite morphological changes. This study shows that the aging coccoid form of H. pylori, although reportedly non-culturable in vitro, remains genetically unchanged indicating that it is likely to be viable.     

Comparison of karyotype analysis and RT-PCR for AML1/ETO in 204 unselected patients with AML

Clinical isolates of Microsporum canis and M. gypseum from humans, dogs and cats were examined by random amplification of polymorphic DNA (RAPD) and Southern hybridization analyses. The RAPD band patterns of six clinical isolates of M. canis were identical to those of standard strains of Arthroderma otae. Of nine clinical isolates of M. gypseum seven and two isolates showed RAPD patterns identical to those of standard strains of A. gypseum and A. incurvatum respectively. Southern blot analysis using a probe (C3) obtained from A. otae DNA revealed that six clinical isolates of M. canis showed specific bands identical to those detected in the standard strains of A. otae. Of nine clinical isolates of M. gypseum, seven and two isolates showed bands hybridized by the C3 probe identical to those detected in A. gypseum and A. incurvatum respectively. Furthermore, the results from mating experiments on these nine clinical isolates of M. gypseum showed complete agreement with the results from RAPD and Southern hybridization analyses. These findings clearly indicate that RAPD and Southern hybridization analyses are very useful in the identification of clinical isolates of M. canis and M. gypseum.     

Ascaris suum: a revision of its early migratory path and implications for human ascariasis

The genetic diversity in a group of Escherichia coli strains belonging to serogroup O6 but expressing different H antigens was investigated by random amplification of polymorphic DNA (RAPD). Isolates of serotypes H16, H1, H31, and non-motile (NM) strains were typed using a set of 3 primers with different G + C contents. The amplified band arrays allowed the identification of 3 main clonal clusters corresponding to each O:H serotype analyzed. Based on their RAPD profiles NM strains could be assigned to either H1 or H31 serotypes. The results indicate that the flagellar antigen and the RAPD fingerprint represent reliable clonal markers in this E. coli group.     

Variability of mitochondrial subgenomic molecules in the meristematic cells of higher plants

MtDNAs from BY-2 cells and rice root were analyzed by random amplified polymorphic DNA (RAPD) assay and Southern hybridization analysis. A number of differences were observed in the RAPD patterns amplified from mtDNAs sampled at different phases of the BY-2 cell culture. RAPD fragments also varied with the template DNAs derived from various areas of rice root tip. When a RAPD fragment was hybridized to restriction fragments of whole DNAs, isolated from the distal area of the apical meristem and differentiated elongation zone of a root, two distinct stoichiometric differences were observed in the hybridization signals. This suggests that the organization of mt-genome in prototypic cells in the root apical meristem differs from that found in the differentiated cells.     

Splenectomy for haematological disease

Five strains of Xanthomonas albilineans, causal agent of leaf scald disease in sugarcane from various geographical regions, were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the DNA level. CsCl-purified genomic DNA from these strains were amplified by the polymerase chain reaction (PCR) using arbitrary 10-mer primers according to standard RAPD conditions and the amplification product profiles analysed by conventional agarose gel electrophoresis. Although most RAPD markers were common to all five strains, unique profiles for each strain were discernible using four 10-mer arbitrary primers individually. Reproducible DNA fingerprints indicate that RAPD analysis can be used to identify and differentiate the X. albilineans strains. This technique has the potential for use in monitoring the appearance of foreign strains of X. albilineans in various geographical regions and could be used for the construction of phylogenetic trees.     

Randomly amplified polymorphic DNAs assess recombination following an induced parasexual cycle in Penicillium roqueforti

Random amplified polymorphic DNAs (RAPDs) were used as a genetic marker system to characterize recombinant strains following the parasexual cycle of Penicillium roqueforti. After protoplast fusion and haploidization of diploid hybrids, segregants characterized by a reassortment of the parental genetic markers displayed specific RAPD fingerprints. The appearance or the loss of RAPD fragments demonstrate that these markers provide an efficient method to analyze recombination and to characterize somatic hybrids.     

Application of DNA fingerprinting to obstetrics and gynecology

Restriction fragment length polymorphisms (RFLP) are variations in the size of restriction fragments of genomic DNA that hybridize to specific probes. They are the consequence of changes in primary DNA sequences, most of which result from small-scale changes in DNA. Recently minisatellite DNA probes that detect many regions of great variability within the human genome have been described. Minisatellite probes consist of multiple repeated copies of a common 10-15 base pair core sequence. On hybridization to restriction enzyme digests of human DNA, they simultaneously detect many highly polymorphic minisatellites at different loci in the genome, and produce band patterns that are individual specific. The band patterns are called "DNA fingerprints" or "DNA barcode" which can be used for individual identification on forensic and legal medicine. In addition to forensic and legal medicine, DNA fingerprinting can be used in both basic research and clinical examination of obstetrics and gynecology. The RFLP bands in DNA fingerprinting are inherited as single Mendelian co-dominants and we can use such minisatellite DNA probe for the determination of zygosity in multiple pregnancy. This probe can be used for the determination of androgenesis as a cause of complete hydatidiform mole. Each polymorphic band in molar tissues could be identified as being of paternal but not maternal origin. Some polymorphic bands of paternal origin were not observed in molar tissues, indicating that endoreduplication of a normal haploid sperm or fertilization by dispermy to an anuclear oocyte with no effective genome could be the cause of complete hydatidiform mole (androgenesis).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene transfer via pollen-tube pathway for anti-fusarium wilt in watermelon

In order to obtain transgenic fusarium wilt resistant watermelon plants, squash DNA was introduced into the ovaries of watermelon plants via the pollen-tube pathway. The introduction of foreign genes into ovaries was accomplished using co-transformation with the CaMV35S-GUS as a marker. Transformed watermelon plants contained integrated copies of the GUS activity and the seeds of transformed progeny produced a blue color when stained with 5-bromo-4-chloro-3-indolyl glucuronide, whereas seeds from untransformed control plants did not. Of 200 transformed seedlings, ten were wilt resistant. The presence of the GUS activity in the genome of stable transgenic seedlings was confirmed by Southern blot analysis. Furthermore, the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with embedded restriction sites showed amplification products unique to these transgenic plants. Primers OPA-1 and OPA-9 gave distinct band patterns of genomic DNA using the polymerase chain reaction.     

Application of random amplified polymorphic DNA analysis for detection of Salmonella spp. in foods

The random amplified polymorphic DNA (RAPD) band patterns from 23 Salmonella spp. produced by use of an oligonucleotide primer (called du primer) designed on the basis of the N-terminal sequence of dulcitol 1-phosphate dehydrogenase (5'-GTGGTGACCCAGGATGGCCAGGTG-3') were different from those from 16 non-Salmonella spp. The bands at 460 and 700 bp were produced in all Salmonella strains tested. These RAPD fragments obtained from Salmonella typhimurium strongly hybridized with the corresponding RAPD bands from the other strains of Salmonella, but not with those from non-Salmonella spp. in Southern blot analysis. The RAPD bands were detected by ethidium bromide staining even when genomic DNA prepared from as few as 2.8 x 10(3) cells was used. The minimum detectable cell number in the initial inoculum of S. typhimurium was 4 x 10(-1) CFU/25 g of raw beef after the preenrichment in Enterobacteriaceae enrichment mannitol (EEM) broth for 6 h and the selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin (DMPBN) medium for 18 h at 42 degrees C. Seven raw foods inoculated with S. typhimurium at numbers from 4 x 10(-1) to 2.6 x 10(2) CFU/25 g of food were positive in both the RAPD analysis and the conventional culture method.     

DNA evidence of the origin of Varroa jacobsoni Oudemans in the Americas

Randomly amplified polymorphic DNA (RAPD) was used to examine possible origin of Varroa jacobsoni Oudemans in the Americas. Among 64 primers screened, 2 primers provided variation which was informative for this study. All V. jacobsoni collected from the United States had the same banding pattern to that of mites collected from Russia, Morocco, Germany, Italy, Spain, and Portugal (Russian pattern). This banding pattern was different from the pattern found for mites collected from Japan, Brazil, and Puerto Rico (Japanese pattern). The Japanese pattern lacked a 766-bp band found in the Russian pattern (OPE-07). With primer OPP-03, the Russian pattern had a distinct band at 442 bp not found in the Japanese pattern. Two bands located at 675 and 412 bp were specific to the Japanese pattern. These results suggest that the V. jacobsoni of the United States is probably predominantly Russian in origin (via Europe), while the V. jacobsoni of Brazil and Puerto Rico are probably predominantly Japanese in origin.