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At least 1,117 records · Page 62Linked to original sources
PubMed2004

Simultaneous infection with multiple strains of Mycobacterium tuberculosis identified by restriction fragment length polymorphism analysis.

Simultaneous infection with multiple strains of Mycobacterium tuberculosis was confirmed in two isolates of a total of 543 culture-positive tuberculosis patients from the Model DOTS area of Thiruvallur, South India, using IS6110 and direct repeat restriction fragment length polymorphism (DR-RFLP). The two isolates showing different intensities of IS6110 band patterns were further analysed by repeating RFLP on their alternate cultures and individual colonies. The results showed respectively two and three distinct IS6110 RFLP patterns, indicating mixed infection. The possibility of cross contamination at laboratory or DNA level was ruled out. Additional DR-RFLP also confirmed infection with multiple strains. This study found evidence of co-infection with two or three different strains of M. tuberculosis, showing distinct RFLP patterns.

Adult↗
PubMed2004

[Techniques and possibilities of traceability of food: genotyping of the domestic animal population as an innovative contribution to food safety].

Traceability of meat has become a very important aspect of quality assurance of food. DNA analyses could be used for identification and verification of farm animals and animal derived products. A prerequisite is the collection of qualified samples from entire populations of production animals or from regionally or specially characterised animal populations. The expenditure for conventional carrying out collection, preservation, cataloguing, and storage would be enormous. Therefore we have developed a simple, reliable, and inexpensive method for the collection using the ear tagging process and for preservation of samples at room temperature. A similar collection technology can also be used for sampling of carcasses, meat and meat products. Isolation of DNA from these tissue samples can be preformed using a new single step technology. For identifying individuals microsatellites and single nucleotide polymorphisms are analysed. Comparison of DNA fingerprints or SNP signatures allows to traceback samples collected from products to the animals they are coming from. If the system will be established on a nationwide basis the total costs would be less than 0.05 EUR per kilogram meat sold.

Animal Identification Systems↗
PubMed1992

The effects of plant polysaccharides and buffer additives on PCR.

A survey of the inhibitory effects of various plant polysaccharides on PCR amplification of a 974-bp section of rbcL in spinach revealed that most of the polysaccharides tested (arabinogalactan, carrageenan, dextran, gum guar, gum karaya, gum locust bean, inulin, mannan, pectin, starch and xylan) were not inhibitory. In contrast, two of the acidic polysaccharides (dextran sulfate and gum ghatti) were inhibitory. The addition of 0.5% Tween 20 reversed the inhibitory effects of gum ghatti (polysaccharide:DNA ratio of 500:1). The inhibitory effect of dextran sulfate (50:1) could be reversed by the addition of Tween 20 (0.25% or 0.5%), DMSO (5%) or polyethylene glycol 400 (5%), but none of these three additives were effective at a 100:1 ratio of dextran sulfate/DNA.

Biotechnology↗
PubMed2001

Extraction and PCR analysis of soy DNA in chocolate.

The manner in which to discriminate between genetically modified (GM) versus non-modified foodstuffs is based on the presence of newly introduced genes at the protein or DNA level. Current available methods are almost exclusively based on the polymerase chain reaction (PCR). These methods consist of three steps: DNA isolation, amplification of the desired DNA fragment and visualisation of the obtained amplification products. The first and crucial step is the DNA isolation. In this study three different methods are described for the isolation of DNA from chocolate, two of which are commercial kits. The results indicate that both kits, in contrast with the non-commercial method, are suitable for the isolation of DNA from chocolate, provided that the adapted PCR conditions are applied.

Cacao↗
PubMed1975

[Spectral and photometric characteristics of different batches of auramine O (OO)].

The spectra of absorption and fluorescence of Auramine O (OO) specimens in water solution and in cells, stained with Schiff-type auramine-SO2 reagents for DNA, were investigated. Using different specimens of the stain the accuracy and sensitivity of the cytofluorometric method were estimated by the next parameters: the value of the useful signal from the nucleus (no), the value of the production of no by the time constant of fading (tau), the value of proportion of the signal to the background (no/nphi) and, in addition, the value of the variation coefficient under the measurement of the rat liver tetraploid cells. The purity of the dye was shown to interfere largely with accuracy and sensitivity of the above method. Auramine 00 of the Reanal production appeared to have the best photometricl characteristics. For this, the maxima of the absorption spectrum in the 320--700 nm region in the solution and in the cell were 371 and 433 nm, and 375 and 436 nm, resp. The maximum of the fluorescence spectrum was found 521 nm and 526 nm, resp. for the solution (0.02%) and for the Auramine-SO2 treated cells. Moreover, an isomer of Auramine O (00) -- Auramine G can be used for detection and photometrical measurement of DNA and glycogen after the Feulgen and PAS reactions, resp.

Aniline Compounds↗
PubMed1984

The use of antibodies to 5-bromo-2'-deoxyuridine for the isolation of DNA sequences containing excision-repair sites.

We have developed an immunological method for isolation and identification of DNA sequences containing 5-bromo-2'-deoxyuridine (BrdUrd) incorporated during UV-induced excision-repair synthesis. DNA fragments containing BrdUrd incorporated during repair synthesis were incubated with goat anti-BrdUrd and rabbit anti-goat IgG, and the antibody-DNA complexes were separated from bulk DNA by nitrocellulose filter binding. With this method, 80% of DNA sequences containing BrdUrd-labeled excision-repair sites were recovered, contaminated with less than 1% of DNA fragments devoid of excision-repair sites. Recovery of DNA fragments containing repair sites was independent of size from 2 to 20 kilobases. We have used this method in conjunction with blot hybridization to demonstrate that repair synthesis occurs in human ribosomal gene sequences in cells treated with UV.

Antibodies↗
PubMed1997

[Immunoglobulin G monoclonal human anti-rhesus Rho(D) to prevent rhesus-incompatibility].

RhD immunisation which follows pregnancy can be prevented by administration to the rhesus-negative mother of 20 micrograms anti-D immunoglobulin per 1 ml of D-positive fetal red cells in the maternal circulation. With the aim of substitution of polyclonal anti-D with monoclonal one we developed human-mouse cell lines producing anti-RhD IgG1 by fusing EBV-transformed human immune lymphocytes with murine myeloma. After several clonings and passages the EBV genome was eliminated from the cell lines. The antibodies were purified from culture supernatants using protein A affinity chromatography and tested for sterility, virus contamination, pyrogenecity, toxicity and DNA content. The monoclonals were compared with the standard polyclonal anti-RhD in in vitro and in vivo assays. In antibody-dependent cell cytotoxicity (ADCC) 2 of 4 studied monoclonals promoted greater RBS lysis than polyclonal anti-D at equivalent concentrations. Detection of binding site number of monoclonals anti-D revealed about 10,000/RBS D-determinants on DCe/dce erythrocyte which agrees with the data for polyclonal anti-D. Best in ADCC monoclonal anti-D sharply increased human autologous 51Cr-labelled rbc sensitised in vitro as well as accelerated clearance of RhD RBS from circulation of Rh-negative volunteers injected with 150 micrograms monoclonal anti-D. After clinical trial using of monoclonal anti-D is permitted in Russia.

Blood Group Incompatibility↗
PubMed1999

Inversion of in situ synthesized oligonucleotides: improved reagents for hybridization and primer extension in DNA microarrays.

Oligonucleotides synthesized in array format suffer from contamination by truncated species. We have developed a method to invert DNA molecules in situ after completed synthesis. Reactive functions at the 5'-ends of the oligonucleotides are permitted to react with functions on the support before the 3'-ends are released, in effect reversing the orientation of full-length oligonucleotides, while any 5'-truncated molecules are lost. This strategy serves both to purify in situ synthesized reagents and to reorient the oligonucleotides, causing them to expose free 3'-hydroxyls. In situ inverted oligonucleotides can be used in assays based on DNA polymerase-assisted extension of immobilized primers, and we demonstrate their utility in minisequencing and in pyrosequencing.

Base Sequence↗
PubMed1984

Mycoplasmal ribosomal RNA genes and their use as probes for detection and identification of Mollicutes.

The number and organization of ribosomal RNA (rRNA) genes in the genome of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species were studied by the Southern hybridization technique. Restriction endonuclease-digested DNAs of the organisms were hybridized with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and with a recombinant plasmid pMC5 constructed of pBR325 and an insert containing M. capricolum genes for 23S, 5S and most of the 16S rRNA gene. The hybridization data indicate the presence of only one or two sets of rRNA genes in the mollicutes tested, a number lower than in eubacteria. The rRNA genes in mollicutes appear to be organized as clusters (acting apparently as operons) in the typical prokaryotic fashion, 5'-16S-23S-5S-3'. Despite the marked sequence homology shared by the rRNA operons of the different mollicutes and of E. coli, the operons are not identical. Thus, there is an EcoRI restriction site in the 16S rRNA genes in only 8 of the 13 species tested. The recombinant plasmid pMC5 has provided a sensitive probe for detection and identification of mollicutes in contaminated cell cultures. The purified DNA of the tested cell culture, or its supernatant fluid, was digested by EcoRI, and Southern blot hybridization of the products was performed with nick-translated pMC5. The probe did not hybridize with eukaryotic DNA. Each of the mollicutes species examinated exhibited a species-specific hybridization pattern. The hybridization tests enabled the identification of the four most prevalent mycoplasma contaminants of cell cultures, M. orale, M. hyorhinis, M. arginini and A. laidlawii. The test is capable of detecting 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10(5) mycoplasmas. The possibility of using this approach for detection and identification of noncultivable mycoplasmas in plant and insect tissues is under investigation.

Animals↗
PubMed1993

Detection of Naegleria fowleri cysts in environmental samples by using a DNA probe.

In this study we tried to detect DNA Naegleria fowleri in artificially contaminated environmental samples, with or without sediments, containing 10(4) cysts of this pathogenic amoeba. We used two assays to extract DNA from samples: first, direct DNA extraction, which gave positive results only for water samples without sediment; second, DNA extraction after sample incubation on agar plates, which allowed us to remove amoeba growing out of the sediments, and which gave positive results for all samples, even those initially with sediments (5, 500 or 500 mg). Thus, this molecular identification appears as a powerful tool to investigate N. fowleri growth in environmental samples.

Animals↗
PubMed2003

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg).

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.

Animals↗
PubMed1991

Sequence variants of human papillomavirus type 16 in clinical samples permit verification and extension of epidemiological studies and construction of a phylogenetic tree.

Genomic variability between different viral isolates provides a powerful epidemiological tool for verifying ultrasensitive diagnostic procedures, understanding infectious pathways in individuals and human populations, and studying viral evolution. The potential of this approach has not yet been exploited for the diagnosis of human papillomaviruses (HPVs) like HPV type 16 (HPV-16), which are involved in genital cancer. Toward this end, we amplified by polymerase chain reaction, cloned, and sequenced a 364-bp noncoding segment of the HPV-16 genome from cell lines, cervical biopsy specimens, and cervical smears. The HPV-16 genomes in the cell lines SiHa and CaSki showed an identical point mutation, and in the SiHa cell line it had an additional 38-bp deletion. Only 4 of 22 cervical lesions biopsied from patients at several hospitals in Singapore contained HPV-16 DNA with the prototype sequence, while the DNAs of the other 18 cervical lesions differed by 1 to 10 mutations. This excludes contaminations with cloned HPV-16 DNA as the source of this DNA. To test whether this diversity was a geographic idiosyncrasy, we analyzed 25 cervical biopsy specimens from Brazil. Eight of these contained the prototype sequence, while 17 were mutated. Altogether, 11 genomic variants were found in the Singaporean samples and 12 genomic variants were found in the Brazilian samples, and only 5 of these occurred identically in both cohorts. All variants could be connected to form a phylogenetic tree, with some branches being specific for each cohort. This suggests that the variants did not originate over a short period in the individual patient but, rather, evolved consecutively while spreading throughout humankind.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗