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At least 919 records · Page 51Linked to original sources
PubMed1991

Determination of urinary 5-hydroxyindole-3-acetic acid by high-performance liquid chromatography with electrochemical detection and direct sample injection.

A method for the routine quantitative determination of the major serotonin metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is described. 5-HIAA was analyzed without prior sample cleanup, using an automated high-performance liquid chromatography system with isocratic elution and electrochemical detection (+0.60 V versus a Ag/AgCl reference electrode). The urine samples were mixed with a solution of the internal standard (5-hydroxyindole-3-propionic acid) and centrifuged. The supernatant was transferred to sealed glass vials, and a 2-microliters aliquot was injected directly onto a C18 reversed-phase analytical column, using an automatic sample injector. Samples of urine could be stored for several months at -80 or at +7 degrees C for 2 days without loss of 5-HIAA. However, a gradual decline with time occurred in crude samples stored at room temperature or above, as well as in urine samples diluted with the mobile phase. The detector response was linear in the range of 0-65 mumol/l 5-HIAA, and the intra- and interassay coefficients of variation were about 5 and 7%, respectively (n = 10).

Chromatography, High Pressure Liquid↗
PubMed1989

Application of a biotin-labelled DNA probe to detect Campylobacter.

A procedure for direct detection of Campylobacter in feces has been developed using a biotin-labelled DNA probe. A species-specific probe (Bio-pCJ174 probe) for C. jejuni and a multivalent probe (Bio-pooled probe) for the thermotolerant Campylobacter species were developed with Bio-11-dUTP. Both probes were used to detect Campylobacter in 100 specimens of chicken feces. The coincidence rate of the results obtained by the Bio-pooled probe and cultivation was 90% and that of the Bio-pCJ 174 probe and cultivation was 96%. Identification of isolates was also carried out with both probes.

Animals↗
PubMed1983

Direct visual detection of protein antigen: importance of surface concentration.

A new type of commercially available substrate was used for visualization of antigen binding to antibodies immobilized on the substrate surface. Addition of antigen induced sufficient increase of the protein layer to allow direct visualization, provided that the antibodies immobilized at the surface were immunosorbent isolated. This finding shows the importance of the surface concentration for direct optical visualization of antigen. The results are discussed in relation to previous reports that it is not possible to visualize directly the binding of protein antigen to surface-immobilized antibodies.

Animals↗
PubMed1996

DNA diagnosis of pulmonary infections: particular emphasis on Mycobacterium tuberculosis.

The past two decades have brought significant changes to the clinical laboratory. Microbiologists now have highly sensitive, rapid and specific molecular methods of identifying infectious agents by the direct detection of DNA or RNA sequences unique to a particular organism. Advanced DNA technology such as nucleic-acid hybridization, PCR and DNA fingerprinting have been used in the direct detection of causative organisms in clinical specimens, with resultant benefits such as increased sensitivity and specificity of the diagnostic approach and reduction of turnaround time. This review outlines a brief description of the various DNA diagnostic tools used in the detection of pulmonary infections with emphasis on their applications in the diagnosis of Mycobacterium tuberculosis.

DNA Fingerprinting↗
PubMed2005

Use of a culture-independent molecular method to study the ecology of Yersinia spp. in food.

A culture-independent method for the direct detection in food of Yersinia spp. was developed in this study. It is based on the amplification of a 359 bp PCR product from the RNA polymerase beta-subunit gene (rpoB) and subsequent analysis by denaturing gradient gel electrophoresis (DGGE). Direct detection of Yersinia spp. by PCR-DGGE was carried out in ready-to-eat vegetables and the results compared with the results of the traditional, culture-dependent method. The DGGE profiles were determined to be species-specific. As a matter of fact, Yersinia enterocolitica, Yersinia intermedia, Yersinia frederiskenii and Yersinia kristensenii showed differential migrations in the gels. Moreover, Y. enterocolitica serotypes O:3, O:5 and O:9 were distinguishable, as well. Only for a limited number of traditionally isolated strains, the biochemical and molecular identification agree. In particular, an overestimation of Y. enterocolitica, as determined biochemically, was observed. Finally when the protocol was applied to 27 food samples, a good correlation was obtained when the results of traditional and direct methods were analyzed. The molecular method was able to identify Y. enterocolitica, not detected by plating analysis. However, for 4 samples, that, by plating analysis, were determined to contain Yersinia spp., no PCR product could be obtained after enrichment, probably due to low numbers of target cells, thereby not allowing the possibility to perform DGGE analysis. The protocol described here represents a reliable tool for the detection of Yersinia spp. in food, which can be used to obtain the needed results faster than with traditional culturing methods.

Colony Count, Microbial↗
PubMed1992

Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associated P-gene product by radiolabelling at newly introduced phosphorylation sites.

Hepatitis B virus (HBV) contains a particle-associated DNA polymerase/reverse transcriptase activity encoded by the P (pol) open reading frame. Due to its low abundance, the corresponding protein has so far escaped direct detection and structural analysis. As a first step to overcome these difficulties, a series of recombinant vaccinia viruses was constructed and used for the synthesis in human hepatoma cells of both the authentic full length protein and of its functional domains. Pulse chase experiments demonstrated that the P-proteins had very short half lives in striking contrast to the viral core protein expressed in parallel with the same system. No evidence was obtained for a specific proteolytic processing of the P-protein as occurring with retroviral pol gene products. Overexpression of P-protein by recombinant vaccinia viruses was then employed to develop a highly sensitive detection method based on the in vitro phosphorylation of newly introduced target sites for protein kinase A. The usefulness of this method was demonstrated in the analysis of encapsidated P-gene products that were transiently expressed from an appropriately modified HBV genome. The results obtained indicate that the P-protein acts unprocessed, at least during the initial steps of nucleocapsid assembly and reverse transcription, and that a fraction of the P-protein molecules is linked as such to the viral DNA. Direct detection of the hepadnaviral P-protein by in vitro phosphorylation should greatly facilitate future analyses on P-protein structure and function.

Base Sequence↗
PubMed2001

Direct DNA hybridization detection based on the oligonucleotide-functionalized conductive polymer.

Electrochemical methods for DNA hybridization detection have many advantages that are very fast to detect hybridization and can be directly applied for a portable DNA sensor. In this paper, an electrochemical method to directly detect DNA hybridization was developed on the basis of a new conductive polymer, which was polymerized on the glassy carbon electrode with a terthiophene monomer having a carboxyl group (3'-carboxyl-5,2',5',2"-terthiophene). The ss-DNA probe was made by chemically bonding an amine-linked C6 alkyl group to the 5' terminus of oligonucleotide (19-mer). The probe moiety was immobilized on the polymer through covalent bonding with a catalyst, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. A difference in admittance was observed before and after hybridization as a result of the reduction of the resistance after hybridization. The highest difference in admittance was observed around 1 kHz before and after hybridization. Hybridization amounts of end two-base and center one-base mismatched sequences were obtained only in a 14.3% response when compared to that for the complementary matched sequence.

Base Pair Mismatch↗
PubMed2004

Direct optical detection of singlet oxygen from a single cell.

Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1deltag --> X3sigmag-) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.

Animals↗
PubMed1998

Photoproduction and direct spectral detection of singlet molecular oxygen (1O2) in keratinocytes stained with rose bengal.

In vivo, keratinocyte skin cells are exposed to photooxidative processes, some of which can be mediated by singlet molecular oxygen (1O2), a species that is very difficult to detect spectrally in cells. We photosensitized 1O2 in cultured HaCaT keratinocytes stained with rose bengal (RB) that localizes exclusively inside the keratinocyte hydrophobic regions, as evidenced by strongly red-shifted absorbance and intense fluorescence. We used keratinocytes grown in a monolayer on a plastic coverslip and in suspension. The phosphorescence spectrum (1200-1350 nm) from 1O2 was strongest when the coverslip containing RB-stained keratinocytes was irradiated in air. The spectral intensity decreased when the coverslip was immersed in D2O during irradiation and was almost completely quenched when it was irradiated while immersed in water. Water not only shortens the 1O2 lifetime but also reabsorbs part of the 1O2 phosphorescence, processes that do not occur when 1O2 is produced in a keratinocyte layer exposed to air. Because the RB was inside keratinocytes, singlet oxygen must also be produced inside the keratinocytes. However, the sensitivity to the extracellular environment suggests that most of the detectable 1O2 phosphorescence originates from those 1O2 molecules that escaped from the cell through its membrane into D2O or into the air, where 1O2 has longer lifetimes. Our results confirm directly that 1O2 is indeed photosensitized in living cells by RB. They also suggest that keratinocyte monolayers may be a good cell model to examine in vitro the production of 1O2 by other photosensitizers of environmental and photomedical interest.

Cell Line, Transformed↗
PubMed1988

Diagnosis of familial hypercholesterolaemia using DNA probes for the low-density lipoprotein (LDL) receptor gene.

Familial hypercholesterolaemia (FH) is one of the most commonly inherited diseases. It is characterised by an abnormal LDL receptor resulting in a selective elevation of serum LDL and cholesterol levels. The correlation between FH and premature heart disease means that these patients contribute significantly to the number of individuals presenting with coronary heart disease. In the work described here cDNA probes to LDL-receptor were used to assess the usefulness of recombinant DNA technology to diagnose familial hypercholesterolaemia. A 3' probe to the LDL-receptor which detects a restriction fragment length polymorphism (RFLP) in linkage disequilibrium with normal and mutant LDL-receptor genes, was found to be potentially informative in 20% of the families studied. In addition a 5' probe to the LDL-receptor may be capable of directly detecting mutations in some 6% of families. We suggest that until further work has established other RFLP's or oligonucleotide probes are synthesised to directly detect mutant LDL-receptor genes, recombinant DNA technology is only of limited value for diagnosing familial hypercholesterolaemia.

Cholesterol↗
PubMed1995

Direct carrier detection for severe haemophilia A: application to families with no available affected male.

Haemophiliae A is a common hereditary disorder of blood coagulation resulting from deficiency of factor VIII. Mutation analysis is the factor VIII gene has been hampered by the large size of the gene and the heterogeneity of molecular defects. In severe haemophiliae A, the most efficient methods of screening for point mutations can detect the lesions in 50 percent of cases only; this is explained by the recent finding (5) of an intragenic inversion that disrupts the factor VIII gene. Since this anomaly could not be characterized by these methods, Lakich et al. have also described a Southern blotting assay that allows a direct determination of the mutation. The use of this assay should greatly increase the feasibility and accuracy with which carrier detection and prenatal diagnosis can be made, as illustrated by the analysis of families with no available affected male that we present here.

Adult↗
PubMed2001

Molecular diagnosis of mycobacteria.

Tuberculosis is one of the leading infectious diseases in the world and is responsible for more than 2 million deaths and 8 million new cases annually. Because of the slow growth rate of the causative agent Mycobacterium tuberculosis, isolation, identification, and drug susceptibility testing of this organism and other clinically important mycobacteria can take several weeks or longer. During the past several years, many molecular methods have been developed for direct detection, species identification, and drug susceptibility testing of mycobacteria. These methods can potentially reduce the diagnostic time from weeks to days. Currently, two nucleic acid amplification methods, the Enhanced Mycobacterium tuberculosis Direct Test (Gen-Probe) and the Amplicor Mycobacterium tuberculosis Test (Roche Diagnostic Systems), have been approved by the Food and Drug Administration for direct detection of M. tuberculosis from clinical specimens. PCR-based sequencing has become commonly used to identify many mycobacterial species. DNA probes have been widely used for species determination of the most commonly encountered mycobacteria. High-density oligonucleotide arrays (DNA microarrays) also have been applied to simultaneous species identification and detection of mutations that confer rifampin resistance in mycobacteria.

Drug Resistance, Microbial↗
PubMed1997

Direct ESR detection or peroxynitrite-induced tyrosine-centred protein radicals in human blood plasma.

Peroxynitrite, the reaction product of O2.- and .NO, is a toxic compound involved in several oxidative processes that modify proteins. The mechanisms of these oxidative reactions are not completely understood. In this study, using direct ESR at 37 degrees C, we observed that peroxynitrite induced in human blood plasma a long-lived singlet signal at g = 2.004 arising from proteins. This signal was not due to a specific plasma protein, because several purified proteins were able to form a peroxynitrite-induced g = 2.004 signal, but serum albumin and IgG showed the most intense signals. Hydroxyurea, a tyrosyl radical scavenger, strongly inhibited the signal, and horseradish peroxidase/H2O2, a radical-generating system known to induce tyrosyl radicals, induced a similar signal. Furthermore peptides containing a Tyr in the central portion of the molecule were able to form a stable peroxynitrite-dependent g = 2.004 signal, whereas peptides in which Tyr was substituted with Gly, Trp or Phe and peptides with Tyr at the N-terminus or near the C-terminus did not form radicals that were stable at 37 degrees C. We suggest that Tyr residues are at least the major radical sources of the peroxynitrite-dependent g = 2.004 signal at 37 degrees C in plasma or in isolated proteins. Although significantly enhanced by CO2/bicarbonate, the signal was detectable in whole plasma at relatively high peroxynitrite concentrations (>2 mM) but, after removal of ascorbate or urate or in dialysed plasma, it was detectable at lower concentrations (100-1000 microM). Our results suggest that the major role of ascorbate and urate is to reduce or 'repair' the radical(s) centred on Tyr residues and not to scavenge peroxynitrite (or nitrosoperoxycarbonate, the oxidant formed in CO2-containing fluids). This mechanism of inhibition by plasma antioxidants may be a means of preserving the physiological functions of peroxynitrite.

Amino Acid Sequence↗
PubMed2005

Archaeal communities in High Arctic wetlands at Spitsbergen, Norway (78 degrees N) as characterized by 16S rRNA gene fingerprinting.

Emissions of the greenhouse gas methane from Arctic wetlands have been studied extensively, though little is known about the ecology and community structure of methanogenic archaea that catalyze the methane production. As part of a project addressing microbial transformations of methane in Arctic wetlands, we studied archaeal communities in two wetlands (Solvatnet and Stuphallet) at Spitsbergen, Norway (78 degrees N) during two summer seasons. Directly extracted peat community DNA and enrichment cultures of methanogenic archaea were analyzed by nested PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rRNA gene fragments. Sequences affiliated with Methanomicrobiales, Methanobacteriaceae, Methanosaeta and Group I.3b of the uncultured crenarchaeota were detected at both sites. Sequences affiliated with Methanosarcina were recovered only from the site Solvatnet, while sequences affiliated with the euryarchaeotal clusters Rice Cluster II and Sediment 1 were detected only at the site Stuphallet. The phylogenetic affiliation of the recovered sequences suggested a potential of both hydrogenotrophic and acetoclastic methanogenesis at both sites. At Solvatnet, there were clear temporal trends in the archaeal community structure over the Arctic summer season. The archaeal community composition was significantly affected by factors influencing the activity of the overall bacterial community, as measured by in situ emissions of CO2. Methane emissions at both sites were influenced more by peat temperatures and thaw depth than by the archaeal community structure. Enrichment cultures for methanogenic archaea determined that most of the methanogens detected directly in peat could grow in culture at 10 degrees C. Culture based biases were indicated in later enrichment steps by the abundant growth of a Methanosarcina strain that was not detected directly in peat samples.

Archaea↗
PubMed1996

[Infectious diseases: current diagnostic methods].

A microbiological diagnosis is either based on the direct detection of the pathogen or on the reaction of the host (indirect method). Many pathogens, in particular small bacteria and viruses, are undetectable by culture or difficult to cultivate. Molecular methods for the direct detection of such pathogens have been widely introduced in many microbiological laboratories within the past few years. The characteristics of these methods as well as new culture methods and new serological tests are discussed.

Bacteria↗
PubMed1994

New technologies in the diagnosis of tuberculosis.

Classic methods for laboratory diagnosis of tuberculosis are time consuming, and resulting delays can adversely affect patient care and tuberculosis control. Newer radiometric culture methods can significantly reduce the time required for detection of Mycobacterium tuberculosis. DNA probe assays and high-performance liquid chromatography of mycolic acids allow identification of isolates in a few hours, and use of these methods is strongly encouraged. A new generation of rapid methods based on techniques of molecular biology will eventually allow direct detection and identification of mycobacteria in clinical samples. These methods use rapid nucleic acid amplification techniques, such as the polymerase chain reaction (PCR), rather than growth of cultures to increase the sensitivity of detection. Assays of this type are currently being evaluated in formal trails. Recent analysis of the mechanisms of drug resistance in M tuberculosis may allow the development of assays for direct detection of drug resistant strains at the genetic level. A luciferase reporter mycobacteriophage assay offers an alternate means for rapidly assessing drug resistance. This assay measures the activity of the drug rather than the genetic basis for resistance. DNA fingerprinting methods have provided the means for distinguishing strains for epidemiological purposes. These new approaches will have a dramatic impact on our ability to rapidly and accurately diagnose tuberculosis.

Antitubercular Agents↗
PubMed2002

Characterization of silicate monomer with sodium, calcium and strontium but not with lithium and magnesium ions by fast atom bombardment mass spectrometry.

Fast atom bombardment mass spectrometry (FABMS) was applied to the direct detection of silica species dissolved in LiCl, NaCl, MgCl(2), CaCl(2) and SrCl(2) solutions in order to investigate its dissolution process in solution. Several species of dissolved silicate complexes in the solution were directly detected by FABMS. The peak intensities of [SiO(2)(OH)(2)Na](-), [SiO(3)(OH)Ca](-) and [SiO(3)(OH)Sr](-) increased with increasing concentrations of NaCl, CaCl(2) and SrCl(2), whereas the peak intensities of [SiO(2)(OH)(2)Li](-) and [SiO(3)(OH)Mg](-) did not increase with increasing concentrations of LiCl and MgCl(2). These results indicte that silicate and cation bind in the solution not after but before ionization. The isotope pattern of Sr(2+) confirmed the existence of the silicate-Sr complex not only with increase of the concentration of silica but also the mass numbers of Sr. The silicate complexes formed with Na(+), Ca(2+) and Sr(2+) showed high stability in chloride solution. This is in good accordance with the fact that Na(+), Ca(2+) and Sr(2+) accelerate the dissolution of silica to form complexes during solution equilibrium. Considering that the stability constant was examined and reported in other papers, this new findings that Mg(2+) does not form a complex with silicic acid (Si(OH)(4)) is very important.

Journal Article↗
PubMed2005

[Diagnosis of urogenital infection by Chlamydia trachomatis. Contribution of genetic amplification techniques].

The majority of patients with Chlamydia trachomatis infection are not aware of ther infection because they do not have symptoms. Therefore, infected individuals may not be identifiable, and chlamydial infection in men may persis for long periods, and can lead to complications such as epididymitis and prostatis. The large group of asymptomatically infected patients is not only at risk of long-term sequelate but also sustains transmission within communities. In asymptomatic and in chronic or persistent chlamydial infections, the level of Chlamydia is very low, and consequently chlamydial infections have never been easy to diagnose. The diagnosis may be based on cell culture, direct detection bacterial antigens, the nucleic acid amplification tests (NAATs) which have become the method of choice, and on the evaluation of antibody titers against various antigenic constituents. Both systemic and local antibodies in secretions can be detected in C. trachomatis infection. The introduction of assays based on amplification of genetic material has subsequently increased the sensitivity of detecting chlamydial infections and offer the opportunity to use non invasive specimens such as first void urine and semen to screen infections either in asymptomatic subjects or male partners of infertile couples. Cell culture or direct detection of bacterial antigens cannot be used for semen and urine samples and are not sensitive enough to rule out infections. Advantages of NAATs are the ability to detect even a small amount of organisms. This enables a high detection rate for C. trachomatis in symptomatic patients, in asymptomatic individuals with a low number of elementary bodies, and diagnosis of persistent infections.

Chlamydia Infections↗