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At least 91 records · Page 5Linked to original sources
PubMed1994

Chemiluminescent microwell hybridization assay for direct detection of human parvovirus B19 DNA.

A method for the direct detection of human parvovirus DNA in serum samples that uses a digoxigenin-labeled RNA probe to hybridize with target B19 DNA, followed by capture of the hybrid onto a microtiter plate wells previously coated with a second oligonucleotide probe was developed. The captured hybrid is then detected with anti-digoxigenin-alkaline phosphatase conjugate and chemiluminescent substrate and the reaction read on a scintillation counter. The relative sensitivities of the microwell and standard dot blot hybridization assays were compared. The chemiluminescent microwell hybridization assay was more sensitive than dot-blot hybridization and could be performed in a few hours. This format, therefore, permits rapid and sensitive detection of parvovirus DNA suitable for the clinical setting.

Base Sequence↗
PubMed2001

Highly sensitive and direct detection of DNA fragments using a laser-induced capillary vibration effect.

A pulsed laser-induced stationary wave capillary vibration detection method was applied to the sensitive detection of capillary gel electrophoresis, and the direct detection of non-labeled nucleic acids, such as DNA sequencing products, was demonstrated. An excimer laser operating at 248 nm was used as a CVL excitation source, and polynucleotides were sensitively detected without derivatization. From an investigation on the endurance of several matrixes to pulsed laser irradiation, a polyacrylamide without a cross-linker (0%C) was found to have adequate endurance, and it exhibited no serious damage during an analysis. A cytosine-terminated sequence reaction product was detected with a sensitivity close to that of laser-induced fluorometry (LIF). These results suggest the feasibility of the highly sensitive detection of ultramicro amounts of biological materials without a pre- or post-column derivatization, which has usually been required in sensitive detection procedures, such as LIF. Furthermore, the feasibility of a novel DNA sequencing method is also suggested.

Acrylic Resins↗
PubMedDate not supplied

Polymerase chain reaction amplification for direct detection of chicken anemia virus DNA in tissues and sera.

Direct detection of chicken anemia virus (CAV) DNA in tissues and sera was investigated by a polymerase chain reaction (PCR) assay. Using a pair of primers constructed to amplify the coding sequence of the CAV DNA genome, the PCR assay was shown to be extremely sensitive, being able to detect 1 fg of CAV replicative form DNA. The oligonucleotide primers used for the PCR yielded 583 base-pair (bp) amplified product, which was sized by ethidium bromide-agarose gel electrophoresis. Tissue samples from seven cases of suspected chicken infectious anemia were obtained for CAV isolation. DNA extracted from the homogenized suspension of pooled tissues of each case was analyzed by the PCR assay. Results showed that five of the seven cases were positive for CAV DNA by PCR, whereas CAV was isolated from four cases only. The PCR assay also detected CAV DNA in two of 37 serum samples from disease-free chickens. The specificity of PCR was confirmed by chemiluminescence dot-blot analysis of the amplified products.

Anemia↗
PubMed2004

Rapid direct detection of multiple rifampin and isoniazid resistance mutations in Mycobacterium tuberculosis in respiratory samples by real-time PCR.

Rapid detection of resistance in Mycobacterium tuberculosis can optimize the efficacy of antituberculous therapy and control the transmission of resistant M. tuberculosis strains. Real-time PCR has minimized the time required to obtain the susceptibility pattern of M. tuberculosis strains, but little effort has been made to adapt this rapid technique to the direct detection of resistance from clinical samples. In this study, we adapted and evaluated a real-time PCR design for direct detection of resistance mutations in clinical respiratory samples. The real-time PCR was evaluated with (i) 11 clinical respiratory samples harboring bacilli resistant to isoniazid (INH) and/or rifampin (RIF), (ii) 10 culture-negative sputa spiked with a set of strains encoding 14 different resistance mutations in 10 independent codons, and (iii) 16 sputa harboring susceptible strains. The results obtained with this real-time PCR design completely agreed with DNA sequencing data. In all sputa harboring resistant M. tuberculosis strains, the mutation encoding resistance was successfully detected. No mutation was detected in any of the susceptible sputa. The test was applied only to smear-positive specimens and succeeded in detecting a bacterial load equivalent to 10(3) CFU/ml in sputum samples (10 acid-fast bacilli/line). The analytical specificity of this method was proved with a set of 14 different non-M. tuberculosis bacteria. This real-time PCR design is an adequate method for the specific and rapid detection of RIF and INH resistance in smear-positive clinical respiratory samples.

Antibiotics, Antitubercular↗
PubMed1995

Mutations in human cytomegalovirus UL97 gene confer clinical resistance to ganciclovir and can be detected directly in patient plasma.

Specific mutations in the UL97 region of human cytomegalovirus (HCMV) have been found to confer resistance to laboratory-adapted strains subjected to ganciclovir selection. In this study, mutations in the UL97 region of HCMV isolates obtained from patients receiving ganciclovir therapy were examined to determine whether they would confer ganciclovir resistance, and if these mutations could be detected directly in the plasma of AIDS patients with progressive HCMV disease despite ganciclovir treatment. A single nucleotide change within a conserved region of UL97 was found in five resistant isolates, resulting in an amino acid substitution in residue 595: from leucine to phenylalanine in one, and from leucine to serine in four resistant isolates. A sixth resistant isolate demonstrated a single nucleotide change, leading to a threonine to isoleucine substitution in residue 659. The role of the 595 amino acid substitution in conferring ganciclovir resistance was confirmed by marker transfer experiments. In further studies, direct sequencing of HCMV DNA present in plasma obtained from persons with resistant viruses revealed the identical amino acid substitutions in plasma as those present in the cultured viruses. These findings indicate that clinical resistance to ganciclovir can result from specific point mutations in the UL97 gene, and that the emergence of the resistant genotype can be detected directly in patient plasma.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Synthesis of a chemiluminescent acridinium hydroxylamine (AHA) for the direct detection of abasic sites in DNA.

[structure: see text] The synthesis of a chemiluminescent acridinium hydroxylamine (AHA) for the direct detection of abasic sites in damaged nucleic acids is described. The reagent reacts readily with abasic sites of damaged calf thymus DNA generated in a time-dependent manner under acid/heat depurination conditions. Preliminary results indicate the sensitivity of the direct chemiluminescent detection format is approximately 0.1 abasic sites detected per 10(6) nucleotides using as little as 200 ng of DNA.

Acridines↗
PubMed2001

Direct detection of low-concentration NO in physiological solutions by a new GaAs-based sensor.

Nitric oxide (NO) acts as a signal molecule in the nervous system, as a defense against infections, as a regulator of blood pressure, and as a gate keeper of blood flow to different organs. In vivo, it is thought to have a lifetime of a few seconds. Therefore, its direct detection at low concentrations is difficult. We report on a new type of hybrid, organic-semiconductor, electronic sensor that makes detection of nitric oxide in physiological solution possible. The mode of action of the device is described to explain how its electrical resistivity changes as a result of NO binding to a layer of native hemin molecules. These molecules are self-assembled on a GaAs surface to which they are attached through a carboxylate binding group. The new sensor provides a fast and simple method for directly detecting NO at concentrations down to 1 microM in physiological aqueous (pH=7.4) solution at room temperature.

Algorithms↗
PubMed2006

Direct detection of double-stranded DNA: Molecular methods and applications for DNA diagnostics.

Methodologies to detect DNA sequences with high sensitivity and specificity have tremendous potential as molecular diagnostic agents. Most current methods exploit the ability of single-stranded DNA (ssDNA) to base pair with high specificity to a complementary molecule. However, recent advances in robust techniques for recognition of DNA in the major and minor groove have made possible the direct detection of double-stranded DNA (dsDNA), without the need for denaturation, renaturation, or hybridization. This review will describe the progress in adapting polyamides, triplex DNA, and engineered zinc finger DNA-binding proteins as dsDNA diagnostic systems. In particular, the sequence-enabled reassembly (SEER) method, involving the use of custom zinc finger proteins, offers the potential for direct detection of dsDNA in cells, with implications for cell-based diagnostics and therapeutics.

Amino Acid Sequence↗
PubMedDate not supplied

Evaluation of a rapid latex agglutination test (Directigen) for the direct detection of group A streptococci from throat swabs.

A rapid group A beta-hemolytic streptococci (GAS) antigen detection test using latex agglutination (Directigen, Becton Dickinson) was compared with a conventional culture method for the direct detection of GAS from throat swabs. One throat specimen was collected from each of 229 patients. After standard inoculation onto sheep blood agar plates, all swabs were tested for GAS antigen. Of the 229 specimens tested, 46 (20%) were GAS-positive by culture. Direct latex agglutination agreed with culture in 222 of them (97%). The sensitivity of the test was 93% (43/46), the specificity 98% (179/183), the positive predictive value 91%, and the negative predictive value 98%. The detection of GAS antigen by coagglutination (Phadebact, Pharmacia) was also carried out. 81% (35/43) of the cultures and Directigen-positive specimens gave a positive coagglutination for GAS. 100% (179/179) of the cultures and Directigen-negative specimens were also negative by the coagglutination test. We conclude that the Directigen rapid latex agglutination test for the direct detection of GAS from throat swabs compares favorably with culture and has the advantage of providing same-day results.

Humans↗
PubMed2003

Electrochemical transducers based on surfactant bilayers for the direct detection of affinity interactions.

The simple methods for the preparing of direct affinity sensors are proposed. The proposed method consists of the immobilizations of either oligonucleotide or antibodies as recognizing elements onto the surfactant bilayer. For DNA-sensor we propose to immobilize oligonucleotide by spontaneous infiltration of hydrocarbon chain bound to oligonucleotide pentadecathymidylate (dT(15)) into the hydrophobic region of surfactant bilayer. The adsorption of antibodies on bilayer surface has resulted in immunosensor development. The direct detection of affinity interactions in both cases has been investigated by impedance spectroscopy. At both studies the significant changes in impedance spectra have observed. The dynamics of response manifestation have been followed the specific DNA-coupling causing the decrease of real part of impedance, whereas the antibody-antigen interaction caused the increase of real part. The obtained results are promising for the development of impedimetric affinity sensors for clinical or environmental applications.

Antigens↗
PubMed2004

Direct-detected rapid-scan EPR at 250 MHz.

EPR spectra at 250 MHz for a single crystal of lithium phthalocyanine (LiPc) in the absence of oxygen and for a deoxygenated aqueous solution of a Nycomed triarylmethyl (trityl-CD3) radical were obtained at scan rates between 1.3 x 10(3) and 3.4 x 10(5)G/s. These scan rates are rapid relative to the reciprocals of the electron spin relaxation times (LiPc: T1 = 3.5 micros and T2 = 2.5 micros; trityl: T1 = 12 micros and T2 = 11.5 micros) and cause characteristic oscillations in the direct-detected absorption spectra. For a given scan rate, shorter values of T2 and increased inhomogeneous broadening cause less deep oscillations that damp out more quickly than for longer T2. There is excellent agreement between experimental and calculated lineshapes and signal amplitudes as a function of radiofrequency magnetic field (B1) and scan rate. When B1 is adjusted for maximum signal amplitude as a function of scan rate, signal intensity for constant number of scans is enhanced by up to a factor of three relative to slow scans. The number of scans that can be averaged in a defined period of time is proportional to the scan rate, which further enhances signal amplitude per unit time. Longer relaxation times cause the maximum signal intensity to occur at slower scan rates. These experiments provide the first systematic characterization of direct-detected rapid-scan EPR signals.

Electron Spin Resonance Spectroscopy↗
PubMed2004

Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein.

The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor. By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants. The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation. The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP. Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM). One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5mM. These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices.

Amino Acid Substitution↗
PubMed1998

Direct detection of eae-positive bacteria in human and veterinary colorectal specimens by PCR.

A PCR test based on the amplification of an eae-specific sequence was designed and evaluated for its ability to directly detect homologous sequences in enteropathogenic Escherichia coli and Citrobacter spp. (amplification of eae open reading frame, 178 bp) in sections of the intestines of humans and animals with colonic lesions. Positive PCR results were observed with eae-positive reference strains of E. coli and Citrobacter rodentium (Citrobacter freundii biotype 4280). Known eae-negative reference strains of E. coli and other laboratory strains of enteric bacteria were negative by the amplification test. The sensitivity of the PCR for detection of eae-positive E. coli and C. rodentium was between 1 and 2 CFU. To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were modified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extraneous bacterial DNA from the PCR amplification cocktail without added template. Sections of colon from three pigs experimentally affected with colon lesions due to enteropathogenic (attaching and effacing) E. coli were PCR positive for bacterial eae genome. Sections from control animals were negative. Sections of colon from one of 18 biopsies from confirmed AIDS patients and from 22 of 35 colorectal cancer patients were PCR positive for bacterial eae genome. The PCR test was a simple and quick method of detecting bacterial eae genome in human and veterinary clinical specimens. This method may remove the need for initial culture and detection of the gene by DNA probing from potential associated lesions. The clear relationship of bacteria containing the eae gene with colonic lesions in the pigs and mice indicates that a similar relationship is possible for human patients having similar lesions.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Laboratory diagnosis of Mycoplasma pneumoniae infection. 2. Comparison of methods for the direct detection of specific antigen or nucleic acid sequences in respiratory exudates.

The efficiency of the direct detection of Mycoplasma pneumoniae in respiratory exudates by an antigen capture, indirect enzyme immunoassay (Ag-EIA), has been compared with its detection with a cDNA probe ('Gen-Probe assay') directed against the specific ribosomal RNA sequences of the organism ('Mycoplasma pneumoniae Rapid Diagnostic System', Gen-Probe, San Diego, California). Both assays showed excellent specificity against a range of mycoplasma species suspended in negative nasopharyngeal aspirates; only M. pneumoniae and M. genitalium reacted. In experiments with graded doses of viable M. pneumoniae cells suspended in negative nasopharyngeal aspirate, the Gen-Probe assay was more sensitive than Ag-EIA; detection limits were respectively 2 X 10(3) c.f.u./ml (3.2 X 10(5) genomes) and 2.5 X 10(4) c.f.u./ml (4 X 10(6) genomes); detection levels 10-100 times less sensitive than culture. The two assays were also tested on nasopharyngeal aspirates or sputum specimens from 90 patients with respiratory infection; 67 of these were culture- or seronegative for M. pneumoniae and 23 were culture- or seropositive. Ag-EIA detected 21 (91%) of the latter but the Gen-Probe assay detected only 5 (22%). Both assays were negative with the 67 culture-/sero-negatives; there were no Gen-Probe assay positive/Ag-EIA negatives. Overall, it is concluded that although Ag-EIA and the Gen-Probe assay are effective substitutes for culture as a diagnostic procedure, there is a significant problem with samples which are culture-negative and from patients who have good serological evidence of current infection. Possible reasons for the disparity between the two assays are advanced.

Antigens, Bacterial↗
PubMed1996

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction.

Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.

Animals↗
PubMed1996

Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and Roche PCR-microwell plate hybridization method (AMPLICOR MYCOBACTERIUM) for direct detection of mycobacteria.

We evaluated the clinical utility of an rRNA amplification-based Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTD) system and a PCR-based Roche AMPLICOR MYCOBACTERIUM system for direct detection of Mycobacterium tuberculosis, M. avium, and M. intracellulare. Of the 422 sputum samples from 170 patients, 137 (121 of M. tuberculosis, 14 of M. avium-M. intracellulare complex [MAC], 2 of mycobacterium other than M. tuberculosis or MAC) were culture positive with the Septi-Chek AFB system. One sample of a contaminated culture results was excluded for further analyses. The AMTD system detected all of the 121 samples which grew M. tuberculosis (sensitivity, 100%). Of the 284 culture negative samples, 28 were positive by this system (specificity, 90.1%). After resolution of the discrepant samples, based on a positive history for culture of the patient, the specificity of this system increased to 99.3%. On the other hand, the AMPLICOR system gave a positive result for 132 out of the 135 culture positive samples for M. tuberculosis or MAC (sensitivity, 97.8%). Of the 284 culture-negative samples, 37 were positive by this system (specificity, 87.0%). The specificity for this system after resolution of the discrepant samples increased to 98.9%. The agreement between the results from the AMTD system and the AMPLICOR system was 98.7%. Both of the systems are highly sensitive and specific for detecting M. tuberculosis and/or MAC directly from sputum samples within hours, and they should be recommended for routine use in the clinical microbiology laboratory.

Bacteriological Techniques↗
PubMed1996

Rapid and high sensitivity test for direct detection of bovine herpesvirus-1 genome in clinical samples.

A procedure for direct detection of the BHV-1 genome in clinical samples, including semen, was developed. The method is based on the PCR amplification of a highly conserved DNA fragment within the glycoprotein gI sequence of the virus (323 bp between nt 1491 to nt 1814). The method is rapid and highly specific for all 27 subtypes assayed, which are included in the clinical and genetically different groups of BHV-1. The viral origin of the PCR product was assessed by Southern hybridization, with an internal probe. The method for DNA isolation from clinical samples included a fast extraction procedure with Chelex 100 resin allowing the loading of larger amounts of DNA in the PCR and in turn increasing the sensitivity of the method of detection. The level of sensitivity achieved by PCR was in the range of 1 TCID(50). This PCR assay may be an useful tool for BHV-1 monitoring in semen banks at low cost.

Animals↗
PubMed1988

Direct detection of HIV-1 RNA from AIDS and ARC patient samples.

Human immunodeficiency virus (HIV), formerly termed human T-lymphotropic virus (HTLVIII/LAV), is the etiological agent of acquired immune deficiency syndrome (AIDS). Direct detection of HIV-1 nucleic acid sequences in patient tissue or blood samples is possible in only a minor fraction of cases due to the low percentage of infected cells (Shaw et al., 1984). We report a modification of the polymerase chain reaction method (PCR) (Saiki et al., 1985), in which we amplify sequences from HIV-1 RNA templates, for the identification of HIV-1 in peripheral blood and tissue samples obtained from AIDS and AIDS-related complex (ARC) patients. This method of HIV-1 detection is at least six orders of magnitude more sensitive than standard nucleic acid detection methods and has direct clinical applications. In vitro tissue culturing of the virus is not required for HIV-1 detection. Using this technique, the sequence in the orfB region of HIV-1 has been amplified and detected from less than 1 microgram of total RNA prepared from a few milliliters of peripheral blood samples. This technique enables the rapid and unambiguous clinical detection of potential HIV-infected individuals and can be used to assay the efficacy of anti-HIV-1 drugs. To enhance the efficiency of this technique, we have appended the prokaryotic T7 RNA polymerase promoter sequence to one of the priming oligonucleotides. After several cycles of PCR with the promoter-containing oligo, a small aliquot of the reaction can be utilized to direct specific and efficient T7 RNA polymerase-mediated transcription of the amplified sequences, thus enhancing the sensitivity and simplifying the labor of the experiment.

AIDS-Related Complex↗