Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Direct detection”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources
PubMed1998

Detection of membrane-bound enzymes in cells using immunoassay and Raman microspectroscopy.

The method of surface-enhanced Raman microspectroscopy was developed for direct detection of membrane-bound enzymes in cells. Cells were cultured, fixed, and incubated with specific primary antibodies and their corresponding labeled secondary antibodies, and surface-enhanced Raman scattering (SERS) was detected directly in the wells of a multiwell plate. First, specific primary antibodies were separately bound to enzymes in cells. Then, the peroxidase-labeled secondary antibodies were added to bind these primary antibodies. Peroxidase substrates, o-phenylenediamine and hydrogen peroxide, were added and reacted for 15 min at room temperature to form azoaniline, a compound with strong Raman scattering. Then, Raman scattering of this enzymatic product was enhanced by silver colloids. Samples were excited with a He/Ne laser at 632.8 nm and SERS was detected by a CCD camera. The SERS spectrum of this product showed an intense peak at 1370 cm-1 and its intensity was used for assessment of cellular enzymes. The observed amount of enzyme was normalized to protein content in each well. The method was successfully used to detect prostaglandin H synthase-1 and -2 (PGHS-1 and -2) in normal human hepatocytes and human hepatocellular carcinoma (HepG2) cells. The detection limit of these PGHS enzymes by this method was about 0.1 pg per well. An immunohistochemical staining was also used to detect the expression of both PGHS isozymes in these cells.

Cell Membrane↗
PubMed2002

Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk.

This work evaluates a newly developed wavelength modulation-based SPR biosensor for the detection of staphylococcal enterotoxin B (SEB) in milk. Two modes of operation of the SPR biosensor are described: direct detection of SEB and sandwich assay. In the sandwich assay detection mode, secondary antibodies are bound to the already captured toxin to amplify sensor response. Samples including SEB in buffer and SEB in milk were analyzed in this work. The SPR biosensor has been shown to be capable of directly detecting concentrations of SEB in buffer as low as 5 ng/ml. In sandwich detection mode, the lowest detection limit was determined to be 0.5 ng/ml for both buffer and milk samples. The reported wavelength modulation-based SPR sensor provides a generic platform which can be tailored for detection of various foodborne pathogens and agents for food analysis and testing.

Animals↗
PubMed1995

A highly sensitive and rapid procedure for direct PCR detection of Leishmania infantum within human peripheral blood mononuclear cells.

We have developed a highly sensitive, simple and rapid procedure to detect Leishmania infantum within human macrophages. It only requires ficoll preparation of peripheral blood mononuclear cells from the patient, and their direct use for Leishmania kDNA amplification by polymerase chain reaction. Under these conditions, about one parasite can be detected in a one million human cell environment. Results, including those of a hybridization step to confirm the diagnosis specificity, are obtained with 24 h, a very short period as compared to current diagnostic methods. This procedure is of particular interest for early detection and early drug treatment of leishmaniasis, especially in the case of HIV coinfection. Furthermore, the method could be useful for monitoring the efficiency of new leishmaniasis treatments in infected patients.

Animals↗
PubMed1995

Monoclonal antibodies for the rapid diagnosis of influenza-B virus infections by ELISA: production and characterization.

BACKGROUND: Monoclonal antibodies directed against conserved epitopes of viral proteins have substantially improved the accuracy of several immunochemical methods in diagnostic virology. OBJECTIVES: To characterize mouse monoclonal antibodies directed against structural protein antigens of influenza-B virus and evaluate their use as diagnostic reagents for the direct detection of such antigens in clinical specimens from patients with respiratory infections of unknown aetiology. STUDY DESIGN: (a) Production and characterization of monoclonal antibodies against influenza-B viral antigens, and (b) their use in two different ELISA systems for detecting influenza-B antigen either directly in clinical specimens or after confirmation by rapid culture in MDCK cells. RESULTS: Four monoclonal antibodies were selected for their specificity for the nucleoprotein antigen as demonstrated by Western blot analysis. The specificity of these antibodies for different epitopes of the nucleoprotein was demonstrated by competition experiments, using unlabelled and biotin-labelled purified antibodies in a sandwich assay. All four antibodies belong to the mouse IgG(2a) isotype, lack haemagglutination inhibition and neutralization properties and exhibit titres as high as 10(-6) in ELISA with as little as 30 ng purified influenza-B virus. ELISA methods using these antibodies detected only influenza-B viral antigens in direct testing of clinical specimens from patients with known influenza-B or influenza-A infections, or after reisolating virus from such specimens in tissue culture of MDCK cells. CONCLUSION: The antibodies were suitable for the direct detection and typing of influenza-B virus in clinical specimens or for use in rapid confirmation cultures.

Journal Article↗
PubMed1993

[Cytomegalovirus infection after transplantation. Virological diagnosis, antiviral treatment].

Cytomegalovirus (CMV) is an important cause of morbidity in organ transplant recipients with two major clinical effects: allograft rejection and pneumonitis. The issue of effective therapy has increased the need for accurate and rapid laboratory methods for diagnosis of viral infections. ELISA, as the most serological sensitive tests, are useful for the identification of active CMV infection, and the serological response can be sometimes detected before viral excretion. There are several commercial reagents for the detection of IgG or IgM CMV antibody, and a great variability in terms of sensibility and specificity. Because of the slow process of isolating CMV in cell cultures, immediate-early antigen detection in infected cells within one or two days of culture, increases twice the sensitivity of viral isolation for leukocyte or bronchoalveolar (BAL) specimens. Differences in sensitivity of the direct detection of CMV antigen in BAL specimens has been reported. Direct detection of CMV antigen in leukocytes is particularly important because CMV viremia is considered to be predictive of significant CMV disease. CMV antigen detection within leucocytes, by immunofluorescence with the aid of monoclonal antibodies to CMV phosphoprotein PP-65, appears to be as specific, more sensitive, and allows a more rapid diagnosis than virus isolation techniques. Some specific CMV probes are now available, but the hybridization techniques involving dot-blot assays of urine or BAL are not enough sensitive to detect small amounts of virus. Closely sensitivity to isolation in culture has nevertheless been reported in the polymorphonuclear fraction of the blood cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Viral↗
PubMed2006

Sensitivity enhancement of liquid chromatographic-direct chemiluminescence detection by on-line post-column solvent mediated pre-oxidative chemiluminescence.

The compatibility of liquid chromatography solvents with oxidizing reagents frequently employed in direct chemiluminescence reactions is examined in this study. Various oxidizing reagents were examined for their response in hydro-organic and micellar mobile phases in both isocratic and gradient elution modes. Mild oxidants like hydrogen peroxide, periodate, cerium and hypochlorite were found to be completely compatible with common reversed phase HPLC solvents posing as no threat to the detection procedure. On the other hand, stronger oxidants like acidic permanganate were found to oxidize organic solvents towards the production of an intense light signal. Although several analytical applications can emerge from this finding, the conjunction of this system with reversed phase HPLC is impractical owing to a significant baseline increase which deteriorates the sensitivity of the analysis. A convenient solution to this problem is proposed based on the regulated on-line post-column pre-oxidation of the organic solvent (SPOC) with mild oxidants that have no influence on the final signal. The analytical utility of this new approach in the determination of organic compounds after chromatographic separation is demonstrated.

Chromatography, High Pressure Liquid↗
PubMed1996

Evolutionary rate variation within Mus APRT.

Rodents are thought to have relatively high rates of evolution, twice as fast as the rates for mammals in other orders. However, the uniformly high rates of evolution inferred for the order Rodentia from Mus musculus and Rattus norvegicus are not consistently found for other rodent species. Using a maximum likelihood phylogenetic algorithm (DNAML), we show here that Mus spicilegus has a fivefold different rate of evolution in 1100 bp around the adenine phosphoribosyltransferase gene (APRT) since its divergence from a common ancestor with Mus musculus. A greater than threefold difference in rates is also found in a comparison of the number of evolutionary events directly detected from the APRT sequences of these two closely related Mus species. The evolutionary events can be directly detected, since M. spicilegus, M. musculus, and the four rodent outgroup species used to determine the ancestral sequence are so closely related. One of the major differences between M. spicilegus and M. musculus that might affect evolutionary rate is the degree of commensalism with man. The Mus species therefore provide a useful model for testing various hypotheses for the causes of rate variations between genes, and possibly, between lineages.

Adenine Phosphoribosyltransferase↗
PubMed2003

[Molecular diagnosis of viral materno-fetal infections].

The main viral infections prenatally detected in fetuses are: cytomegalovirus (CMV), parvovirus B19, rubella virus and varicella-zoster virus infections. Prenatal diagnosis is based on the direct detection of the virus by culture (CMV), of its antigens (CMV) or of its genome, essentially by PCR. This direct detection can be done either on fetal blood or on amniotic fluid. Prenatal diagnosis can also be performed by detection of specific IgM in fetal blood (rubella). Non specific markers of viral infections can also help diagnosis. At the present time, prenatal diagnosis is essentially based on the detection of the viral genome in amniotic fluid. In order to better appreciate the severity of fetal infections, some groups have tried to identify prognostic markers of these infections. The viral load could play a role in certain infections (CMV).

Adult↗
PubMed2002

Photobacterium damselae ssp. piscicida: detection by direct amplification of 16S rRNA gene sequences and genotypic variation as determined by amplified fragment length polymorphism (AFLP).

A PCR protocol for the rapid diagnosis of fish 'pasteurellosis' based on 16S rRNA gene sequences was developed. The procedure combines low annealing temperature that detects low titers of Photobacterium damselae but also related species, and high annealing temperature for the specific identification of P. damselae directly from infected fish. The PCR protocol was validated on 19 piscine isolates of P. damselae ssp. piscicida from different geographic regions (Japan, Italy, Spain, Greece and Israel), on spontaneously infected sea bream Sparus aurata and sea bass Dicentrarchus labrax, and on closely related American Type Culture Collection (ATCC) reference strains. PCR using high annealing temperature (64 degrees C) discriminated between P. damselae and closely related reference strains, including P. histaminum. Sixteen isolates of P. damselae ssp. piscicida, 2 P. damselae ssp. piscicida reference strains and 1 P. damselae ssp. damselae reference strain were subjected to Amplified Fragment Length Polymorphism (AFLP) analysis, and a similarity matrix was produced. Accordingly, the Japanese isolates of P. damselae ssp. piscicida were distinguished from the Mediterranean/European isolates at a cut-off value of 83% similarity. A further subclustering at a cut-off value of 97% allowed discrimination between the Israeli P. damselae ssp. piscicida isolates and the other Mediterranean/European isolates. The combination of PCR direct amplification and AFLP provides a 2-step procedure, where P. damselae is rapidly identified at genus level on the basis of its 16S rRNA gene sequence and then grouped into distinct clusters on the basis of AFLP polymorphisms. The first step of direct amplification is highly sensitive and has immediate practical consequences, offering fish farmers a rapid diagnosis, while the AFLP is more specific and detects intraspecific variation which, in our study, also reflected geographic correspondence. Because of its superior discriminative properties, AFLP can be an important tool for epidemiological and taxonomic studies of this highly homogeneous genus.

Animals↗
PubMed1991

Rapid detection of type A influenza viruses with monoclonal antibodies to the M protein (M1) by enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay.

Monoclonal antibodies (MAbs) to the M protein (M1) were used in the development of direct detection systems for type A influenza viruses in clinical specimens. Optimal detection by an enzyme-linked immunosorbent assay was achieved when MAbs were used as capture antibodies and rabbit polyclonal antibodies were used as sandwich antibodies. Detection by the enzyme-linked immunosorbent assay required amplification of the virus. direct detection in clinical specimens (nasopharyngeal aspirates) was accomplished when MAbs recognizing two distinct antigenic sites of M1 were used in a time-resolved fluoroimmunoassay. Type A influenza viruses could be detected equally well in specimens obtained during epidemics of both H3N2 and H1N1 influenza viruses.

Antibodies, Monoclonal↗
PubMedDate not supplied

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay.

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.

Animals↗
PubMed2005

Rapid-scan EPR with triangular scans and fourier deconvolution to recover the slow-scan spectrum.

Direct-detected rapid-scan EPR signals were recorded using triangular field scan rates between 1.7 and 150 kG/s for deoxygenated samples of lithium phthalocyanine (LiPc) and Nycomed trityl-CD3. These scan rates are rapid relative to the reciprocals of the electron spin relaxation times and cause characteristic oscillations in the signals. Fourier deconvolution with an analytical function permitted recovery of lineshapes that are in good agreement with experimental slow-scan spectra. Unlike slow-scan EPR, direct detection rapid-scan EPR does not use phase sensitive detection and records the absorption signal directly instead of the first derivative of the absorption signal. The amplitude of the signal decreases approximately linearly with applied magnetic field gradient. Images of phantoms constructed from samples of LiPc and trityl-CD3 were reconstructed by filtered back-projection from data sets with a missing angle. The lineshapes in spectral slices from the image are in good agreement with slow-scan spectra and the spacing between sample tubes matches well with the known sample geometry.

Algorithms↗
PubMed1994

Direct PCR detection of foot-and-mouth disease virus.

A PCR assay for the detection and characterization of foot-and-mouth disease virus was developed. The procedure allows RT-PCR amplification following direct adsorption of viral suspensions to microtiter plates, avoiding previous steps of phenol-extraction or heating. Using this procedure, FMDV-specific (based on 3D gene sequences), as well as serotype-specific (based on VP1 gene sequences) amplification were achieved for viral samples of serotypes A, O and C, either from cell culture supernatants or from lesions of infected animals. The assay allowed detection of around 15 PFU, being 500-fold more sensitive than a conventional indirect ELISA. This new method constitutes a simple, rapid and efficient alternative for the diagnosis and characterization of FMDV by PCR.

Animals↗
PubMed1992

Evaluation of four methods for rapid detection of adenovirus.

Four methods for rapid detection of adenovirus were evaluated by testing retrospectively 28 frozen clinical specimens from which an adenovirus strain had been isolated. After thawing all specimens were retested for the presence of adenovirus by conventional culture on KB cells and found to be positive. The four tests used for rapid detection of adenovirus were a 48-hour culture technique, and an immunoassay, a latex agglutination test and an immunofluorescence assay for direct detection of viral antigen using commercially available reagents. Of the 28 specimens all were positive in the 48-hour culture, 25 (89%) positive in the immunoassay and 10 (36%) positive in the latex agglutination test. Six of eight nasopharyngeal aspirate specimens were positive in the immunofluorescence assay. Twenty-five clinical specimens negative for adenovirus on conventional culture were also negative in the 48-hour culture technique. Overall, the rapid (48-hour) culture technique was 100% sensitive and 100% specific compared to conventional culture. The direct detection of viral antigen by immunoassay was less sensitive, however results were available within a few hours. Prospective comparative studies are warranted to determine whether these rapid techniques could replace conventional culture in the routine diagnosis of adenovirus infection.

Adenoviridae↗
PubMedDate not supplied

Chemiluminescent acridan phosphate labelling compounds for detection in gels.

We have developed a chemiluminescent acridan phosphate labelling compound which produces a flash of light upon chemical triggering. Sequential treatment of the label with acid and a strong base, one of which contains a peroxide, initiates light emission as a rapid flash lasting 1--2 s. Labelling of analytes with these compounds permits their direct detection in a non-enzymatic assay format. Representative compounds have been linked to BSA as a model protein analyte and detected by chemiluminescence assay. Light intensity correlated with the amount of BSA over six orders of magnitude, permitting the detection of 50 amol of protein in solution. We have also demonstrated for the first time that labelled proteins can be separated by electrophoresis without destruction of the label and that chemiluminescence can be produced and detected directly in the gel. The ease, speed and sensitivity of detection by this new method should enable the development of simpler assays for a wide variety of analytes.

Acridines↗
PubMed1993

Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis.

A rapid screening test was recently established for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis, a region identified as the locus for rifampin resistance (Rifr). The detection method involved the amplification by polymerase chain reaction (PCR) of the Rifr region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products. Experience using two different PCR-SSCP formats for the evaluation of BACTEC cultures and sputum is presented here; the previously described manual procedure for the detection for the detection of radiolabelled amplification products and an automated SSCP by which fluorescein-labelled products were detected on a Pharmacia DNA sequencer apparatus. All 17 different Rifr mutations known to date were consistently detected. PCR-SSCP could be used for the evaluation of minimally grown cultures (BACTEC 12B medium with a growth index of < or = 100) and for direct screening of microscopically positive sputa with greater than 10 organisms per field (magnification, x250). Implementation of this technique could result in rapid detection of rifampin resistance in M. tuberculosis, a marker of multidrug-resistant tuberculosis.

Base Sequence↗
PubMed2005

[Optical detection system for micro biochemical analyses].

For the need of biochemical chip, which consumes fewer specimens and is easy to integrate with micro-fluid chip, two kinds of spectrophotometric analysis methods are described in the present paper. Both the direct detection method and evanescent wave detection method are used in the experiments with visible light (460-800 nm). The experimental results proved that the direct detection is simple and evident; on the other hand the evanescent wave detection method consumes much less reagent and is easy to integrate with microchips.

Microfluidic Analytical Techniques↗