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Results for “Random Amplified Polymorphic DNA Technique”

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PubMed1995

Variations in DNA concentrations significantly affect the reproducibility of RAPD fingerprint patterns.

The influence of the DNA concentration was tested using two different primers and nine DNA samples. Major modifications in the DNA banding pattern were apparent between successive dilutions. Such differences could be explained by concomitant changes in three different molecular conditions: the presence of perfect priming sites, the amplification of rare sites and the existence of mismatch annealing events. At low DNA concentrations (less than 1 pg/microliter), molecular events occurred at random and had a direct consequence on the reproducibility of RAPD profiles. At the appropriate DNA concentration (between 100 ng/microliters and 10 pg/microliters), reproducibility was adequate at a given concentration, but RAPD profiles differed from one dilution to another. These observations demonstrate the usefulness of the bis-benzimide method for quantification of DNA extracts.

DNA, Bacterial↗

PubMed2004

Molecular epidemiology of Cryptococcus neoformans isolates from AIDS patients of the Brazilian city, Rio de Janeiro.

A high biodiversity of Cryptococcus neoformans isolates is known to exist in some Brazilian urban areas, raising the possibility that patients may encounter multiple inoculum sources in their daily life. C. neoformans isolates from two groups of AIDS patients with cryptococcosis from Rio de Janeiro were studied by polymerase chain reaction (PCR) fingerprinting and randomly amplified polymorphic DNA (RAPD) analysis. The first group contained 60 serial isolates obtained from 19 patients over periods ranging from 18 to 461 days; the intent was to determine whether the original strain persisted or whether reinfection with a new strain occurred. The second group was made up of 22 isolates from 11 patients, and consisted of a pair of isolates collected from blood and cerebrospinal fluid from each patient either before or shortly after treatment was initiated. The aim was to determine if the patient was infected by different strains simultaneously. All isolates were subtyped by PCR fingerprinting, using minisatellite (M13), and microsatellite [(GACA)4 and (GTG)5] specific primers, and RAPD analysis employing the combined primers 5SOR and CN1. The majority of isolates were C. neoformans var. grubii, specifically, molecular types VNI or VNII, but numerous distinguishable subtypes were found. Only three isolates were C. n. var. gattii (molecular types VGI or VGII). Except in two cases, all isolates obtained from the same patient showed identical PCR profiles independent of time of isolation or body site. Almost all patients, however, carried unique genotypes not found in any other patient. Our results confirm that persistent cryptococcal infection is caused by relapse rather than reinfection, but they also show that in exceptional cases, patients may be infected with more than one C. neoformans strain.

AIDS-Related Opportunistic Infections↗

PubMed2005

DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics.

The two most widespread biotypes of Bemisia tabaci (Gennadius) in southern Europe and the Middle East are referred to as the B and Q-type, which are morphologically indistinguishable. In this study various DNA markers have been developed, applied and compared for studying genetic diversity and distribution of the two biotypes. For developing sequence characterized amplified regions (SCAR) and cleaved amplified polymorphic sequences (CAPS) techniques, single random amplified polymorphic DNA (RAPD) fragments of B and Q biotypes, respectively, were used. The CAPS were investigated on the basis of nuclear sodium channel and the mitochondrial cytochrome oxidase I genes (mtCOI) sequences. In general, complete agreement was found between the different markers used. Analysis of field samples collected in Israel for several years, using these markers, indicated that the percentage of the Q biotype tends to increase in field populations as time progresses. This may be attributed to the resistance of the Q biotype to neonicotinoids and pyriproxyfen and the susceptibility of the B biotype to these insecticides.

Animals↗

PubMed2001

Lack of prezygotic isolation by assortative mating between the two cryptic species of the polytypic Schistosoma intercalatum taxon.

Ten mice were individually exposed to 30 male and 30 female cercariae of Schistosoma intercalatum from Cameroon and to 30 male and 30 female cercariae of S. intercalatum from Zaire. Ten weeks later, the nine surviving mice were killed, and a total of 263 adult schistosomes (228 pairs, 7 unpaired males, 28 unpaired females) were perfused. All worms were identified utilising the random amplified polymorphic DNA technique using primer OPG06 which enables the differentiation of the Cameroon and Zaire genotypes. The data demonstrate that there were no significant differences between the number of homospecific pairs observed compared with the expected number under the null hypothesis. Matings occurred in a random manner indicating that mate choice did not exist for the two genotypes of S. intercalatum. Hence there is no prezygotic isolation mechanism which would prevent the interaction of the two genotypes if they were sympatric. However, as they are allopatric, the possibility of the production of unfit hybrids does not arise. Consequently, the reinforcement of a prezygotic isolating mechanism does not play a significant role in the speciation process.

Animals↗

PubMedDate not supplied

Characterization of enterotoxigenic Escherichia coli by random amplification of polymorphic DNA.

Two enterotoxigenic Escherichia coli (ETEC) strains (H10407 and 4011-1) were characterized by random amplification of polymorphic DNA (RAPD) profiles using 10-mer oligonucleotides with diverse GC content. All tested primers yielded arrays of amplified DNA products ranging in size from 200 to 3000 bp. The effects of annealing temperature, template concentration and GC content of the primers were evaluated and an optimal reaction procedure was established. Application of the RAPD analysis to ten ETEC strains belonging to five different serotypes showed that strains of the same serotype shared identical or almost identical band profiles, suggesting a similar genetic composition. The use of RAPD profiles as a tool in epidemiological analysis of ETEC is discussed.

Child↗

PubMed2003

Epidemiology of Candida colonization in an intensive care unit of a teaching hospital in Kuwait.

The incidence of Candida infections in intensive care units (ICU) is increasing. Although most cases of candidemia are caused by commensal strains colonizing the patients' own body sites, recent studies have suggested that the source of Candida infection can also be exogenous. This study was carried out to prospectively investigate the frequency of Candida colonization among patients and health care personnel of an ICU of a teaching hospital in Kuwait. A total of 57 patients and 45 nurses were investigated. Candida isolates were identified to the species level by the Vitek identification system. The typing of selected isolates was performed by randomly amplified polymorphic DNA (RAPD) using three different arbitrary primers (CARAPD1, AP3, CT5). Of the 526 samples collected from 57 patients, 180 (34%) yielded Candida species. These included 112 (62%) C. albicans, 37 (21%) C. glabrata, 15 (8%) C. parapsilosis, 11 (6%) C. tropicalis, 3 (2%) C. krusei and 2 (1%) C. lusitaniae isolates. Thirty-seven patients (65%) were colonized by Candida at some point of stay in the ICU. Seven (12%) patients yielded more than one Candida species from the sites/specimens tested. The frequency of Candida isolation was highest from oropharynx, followed by rectum, groin, urine and trachea. Twenty-five (6%) of the 448 swabs from the nurses yielded Candida and included C. albicans (n = 16), C. parapsilosis (n = 4) and C. famata (n = 5). While all the three primers yielded varying patterns in RAPD analyses for each Candida species, the results obtained by AP3 were most discriminatory. The data showed that the colonizing Candida isolates recovered from various body sites of the patients, as well as the nursing staff, were different. However, when RAPD profiles of three blood culture isolates from candidemic patients were analyzed, the DNA fingerprint produced by one C. parapsilosis blood culture isolate was similar to C. parapsilosis recovered from the hands of two nurses, suggesting exogenous acquisition of infection.

Adolescent↗

PubMed2005

Field and experimental studies on Dicrocoelium dendriticum and dicrocoeliasis in northern Spain.

The transmission, control and the relationship between Dicrocoelium dendriticum and its definitive (sheep and cattle) and intermediate (molluscs and ants) hosts under natural and experimental conditions are described. Eleven species of molluscs and four of ants were found infected with larval D. dendriticum in León province, north-west Spain. Infected ants were observed between April and November and in tetania at 7.5-26.9 degrees C. The highest shedding of eggs by sheep and cattle was detected in winter. Two treatments applied in November and January were the most effective. In experimentally infected molluscs, the parasite was not visible under the stereomicroscope, at least until 50 days post-infection (p.i.). The prepatent period in experimentally infected lambs was 49-79 days p.i. The number of eggs per gram increased with the days p.i. and the parasite burden. The aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, leukocyte and neutrophil values of infected lambs increased, but those of lymphocytes decreased. Using the enzyme-linked immunosorbert assay technique, the IgG antibody response to excretory-secretory and somatic antigens of D. dendriticum was positive from day 30 p.i., although the maximum antibody levels were observed on day 60 p.i. The number of worms per lamb ranged between 30 and 2063. Cholangitis and cholangiectasia of the septal bile and hepatic ducts were observed. The best enzymatic systems for adult and larval D. dendriticum characterization were lactate dehydrogenase, glucose phosphate isomerase and phosphoglucomutase. Genetic variability of adult D. dendriticum was high using the random amplified polymorphic DNA technique.

Animals↗

PubMed1996

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius.

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.

Acid Phosphatase↗

PubMed1997

An alternative approach to evaluating the intraspecific genetic variability of parasites.

Analysis of DNA polymorphisms provides important information for the molecular characterization of parasite strains and clones. Because we still know little about the genomes of parasites, such analysis has to rely on methods applicable to any eukaryotic genome, such as DNA fingerprinting with multilocal minisatellite probes and the polymerase chain reaction (PCR)-based random amplified polymorphic DNA technique (RAPD). However, DNA fingerprinting is cumbersome and needs large amounts of parasite DNA, and RAPD can exhibit low reproducibility and spurious bands, both of which appear to be related to the low stringency of the PCR procedure. Riva Oliveira, Andréa Macedo, Egler Chiari and Sérgio Pena here evaluate the applicability to parasites of a technique described two years ago called simple sequence repeat-anchored PCR amplification (SSR-PCR), in which a single primer is needed [the (CA)(8)RY primer] and highstringency conditions are applied.

Journal Article↗

PubMedDate not supplied

Biodegradation of alkylpyridines by bacteria isolated from a polluted subsurface.

Ten bacterial strains were isolated from alkylpyridine polluted sediments 7.6 m below the surface. These strains were able to degrade 11 different alkylpyridine isomers. Degradation rates depended on number and position of the alkyl group. Isomers with an alkyl group at position 3 were more resistant to microbial attack. Of the 10 strains, 6 isolates were selected for detailed study. These isolates mineralized the isomers to CO2, NH4+, and biomass. All strains were gram-negative rods with a strict aerobic metabolism. Characterization of physiological and biochemical properties revealed similarity between strains. Eeach strain however, had a limited substrate range which enabled it to degrade no more than 2 to 3 compounds of the 14 alkylpyridine isomers tested. Examination of the genetic variability among cultures with the randomly amplified polymorphic DNA technique revealed high levels of genomic DNA polymorphism. The highest similarity between 2 strains (0.653) was observed between 2-picoline and 3-picoline degrading cultures. The molecular basis of the differences in substrate specificity is under investigation.

Bacteria↗

PubMed2005

Avian pathogenic Escherichia coli transmission from broiler breeders to their progeny in an integrated poultry production chain.

Early cases of colibacillosis with omphalitis, yolk sac infection and increased mortality were observed in five broiler chicken flocks (A1, A2, A3, A4 and B1) from two broiler breeder flocks A and B, respectively. Avian pathogenic Escherichia Coli (APEC) serotype O78, Fim/Tsh/Iuc pathotype, were isolated from flocks A, A1, A2, A3 and A4, and APEC serotype O139, pathotype Fim/Iuc, from flocks B and B1. APEC O78 strains isolated from broiler chicks A1, A2, A3 and A4, originating from breeder flock A, had the same antibiotic resistance pattern as APEC O139 strains isolated from broiler chicks B1 and breeder B. The random amplified polymorphic DNA technique performed on APEC strains revealed two distinct clusters of genetic similarity: cluster I consisted of some APEC O78 and cluster II of APEC O139. These results indicated that a transmission of APEC strains from adults A and B to their respective progeny could occur.

Animals↗

PubMed1997

The genetic structure of Escherichia coli populations in feral house mice.

Escherichia coli was isolated from feral house mice (Mus domesticus) during the course of a mouse plague in the state of Victoria, Australia. Two farms were sampled over a period of 7 months and a total of 447 isolates were collected. The isolates were characterized using the techniques of randomly amplified polymorphic DNA and multi-locus enzyme electrophoresis. The mean genetic diversity of this E. coli population (H = 0.24) was found to be substantially lower than the diversity of an E. col population reported elsewhere for a single human host. Analysis of the allozyme data revealed that there were significant differences in the relative abundance of genotypes between the two localities sampled and among sample dates. Overall, however, spatial and temporal effects accounted for less than 5% of the genotypic diversity. Allele frequencies and the relative abundance of the more common genotypes did not differ between male and female hosts. The number of genotypes and genotype diversity increased as the age of the host increased, suggesting that the mice are continuing to acquire new E. coli clones throughout their life. The frequency of some alleles changed with respect to host age, which indicates that clone acquisition may not be a random process. It is argued that the low level of genetic diversity observed in this population of E. coli reflects the boom and bust nature of mouse population density in this region of Australia.

Alleles↗

PubMed2001

The microbial diversity in picoplankton enrichment cultures: a molecular screening of marine isolates.

Picoplankton bacteria from a North Sea water sample were cultured under a variety of different conditions (nutrients, temperature, light, agitation, adhesion). Fluorescent in situ hybridization (FISH) analysis of the enrichments showed complex communities which were dominated by gamma-Proteobacteria or beta-Proteobacteria, followed by alpha-Proteobacteria and bacteria from the Cytophaga/Flavobacterium/Bacteroides (CFB) cluster. Among 410 isolates, a high degree of diversity was found, both with respect to colony color and morphology and with respect to genetic diversity. Isolated bacteria were classified into the main taxa by a special PCR approach, termed signature PCR (SIG-PCR). It was based on an oligo primer mixture targeting 16S rDNA which yielded PCR products of taxon-specific lengths. Again, gamma-Proteobacteria dominated (48%), followed by alpha-Proteobacteria (20%). beta-Proteobacteria were rarely isolated (eight strains of 410). The CFB cluster comprised the second largest phylum (14%), and 7.5% of all isolates belonged to the high-GC Gram-positives. Thus, isolated bacteria were representative of enrichment communities with the exception of the beta-Proteobacteria, which were detected in high abundance in certain enrichments by FISH but not isolated, and the high-GC Gram-positives, which were cultivated but not detected by FISH. A genomic fingerprinting technique, randomly amplified polymorphic DNA, showed that among 58 CFB isolates only 18 identical genotypes were found, and among the 84 alpha-Proteobacteria only eight identical genotypes were present. The data show the enormous diversity of cultivated bacteria from picoplankton enrichment cultures of one North Sea water sample, which is only a small fraction of the total picoplankton community.

Journal Article↗

PubMed2002

Distribution of genes encoding the trypsin-dependent lantibiotic ruminococcin A among bacteria isolated from human fecal microbiota.

Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogenetically related strains that can make up a significant part of the human fecal microbiota.

Bacteria↗

PubMed1997

The first clinical isolate of Legionella parisiensis, from a liver transplant patient with pneumonia.

A bluish white autofluorescent strain of Legionella was isolated from the tracheal aspirate of a female liver transplant patient who developed hospital-acquired pneumonia. This strain had biochemical characteristics compatible with those of L. cherrii, L. anisa, and L. parisiensis and could not be differentiated from L. bozemanii and L. parisiensis by the direct fluorescent-antibody assay. Phylogenetic analysis of partial 16S rRNA gene sequences of this strain (ATCC 700174) revealed the closest homology to the species L. parisiensis (99.5%). An L. parisiensis species-specific profile was also identified by a random amplified polymorphic DNA technique. This is the first report of L. parisiensis isolation from humans.

Adult↗

PubMed2006

Molecular characterization of Haemophilus ducreyi isolates from different geographical locations.

The technique of random amplified polymorphic DNA (RAPD) was adapted and optimized to study Haemophilus ducreyi isolates. A panel of 43 strains isolated from chancroid patients from different countries in Africa, Europe, North America, and Asia were characterized. The strains were also studied with respect to lipooligosaccharide (LOS) migration and immunoblotting patterns and the presence of cytolethal distending toxin genes. The RAPD method with the OPJ20 primer generated nine banding patterns (1 to 9). The majority of the isolates were clustered into two major profiles, 14 and 13 strains into profiles 1 and 2, respectively, and just a few strains revealed patterns 3 and 4. The isolates from Thailand were exceptional in that they showed greater diversity and were represented by six different RAPD patterns, i.e., patterns 3 and 5 to 9. The LOS migration and immunoblotting analyses revealed two different patterns, which indicated long and short forms of LOS; the former was found in 20/23 tested strains. Two strains that expressed the short form of LOS were grouped into RAPD pattern 4. The absence of cdtABC genes was observed in only 4/23 strains, and three of these isolates were assigned to RAPD pattern 4. Our results showed limited genotypic and phenotypic variations among H. ducreyi strains, as supported by the conserved RAPD and LOS profiles shared by the majority of the studied strains. However, the RAPD method identified differences between strains, including those from different geographic areas, which indicate the potential of RAPD as an epidemiological tool for the typing of H. ducreyi isolates in countries where chancroid is endemic.

Bacterial Toxins↗

PubMed2003

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese.

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.

Carbohydrate Metabolism↗

PubMed2001

Genetic diversity of inbred rye lines evaluated by RAPD analysis.

This study presents an attempt to supply breeders of hybrid rye with more genetic information on inbred lines, using molecular markers. Eighteen polymorphic loci detected by means of the RAPD (Random Amplified Polymorphic DNA) technique and mapped on 2R-7R rye chromosomes, were applied to study genetic similarities among forty inbred lines of rye. The lines were grouped in four main clusters revealed on dendrogram, which was generally consistent with the pedigree data. Mapped RAPD markers were shown to be a useful tool for phenetic studies in rye. Additionally, a system of 20 polymorphic fragments, detected by three primers, was developed for fingerprinting of rye lines. The system of RAPD markers, which was developed in this study, should be helpful in characterisation of rye genetic stocks used for breeding.

Journal Article↗