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PubMed2000

Use of polymerase chain reaction (PCR) and DNA probe hybridization to determine the Gram reaction of the infecting bacterium in the intraocular fluids of patients with endophthalmitis.

OBJECTIVES: To evaluate polymerase chain reaction (PCR) combined with DNA probe hybridization to determine the Gram reaction of the bacterium in intraocular specimens from patients with infectious endophthalmitis. METHODS: Fifty-seven intraocular specimens - 17 aqueous humor (AH) and 40 vitreous fluid (VF) - from 55 patients with clinically diagnosed infectious endophthalmitis and 25 control intraocular specimens from non-infectious ocular disorders (10 AH and 15 VF) were evaluated by microscopy, culture and PCR-DNA probe hybridization to detect the Gram reaction of the bacterium. RESULTS: PCR-DNA probe hybridization was specific and sensitive to detect 30 fg of both gram-positive and gram-negative bacterial DNA. None of the controls showed bacteria by microscopy, culture or PCR. Of the 57 intraocular specimens, conventional microbiological methods could detect a bacterial aetiology in 32 (56.1%), while PCR-DNA probe hybridization could detect 52 (91.2%) specimens. This difference was statistically significant (P= 0.003). In bacteriologically positive specimens, there was absolute correlation of the Gram reaction between the results of smear and culture methods and PCR-DNA probe hybridization. Of the 25 bacteriologically negative specimens, 20 (80%) were positive by PCR-DNA probe hybridization, of which seven (35%) were gram-positive, 12 (60%) gram-negative and one (5%) positive by both. Results of PCR on AH and VF were not significantly different. CONCLUSION: PCR and DNA probe hybridization to determine the Gram reaction of the bacterium in intraocular fluids is a specific and sensitive method in the diagnosis of bacterial endophthalmitis. AH is an ideal specimen for PCR, since its collection is a simple and safe office procedure.

Aqueous Humor↗
PubMedDate not supplied

[DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences].

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.

Base Sequence↗
PubMed1986

Detection of bovine herpesvirus-specific nucleic acids by in situ hybridization with biotinylated DNA probes.

Biotin-labeled DNA probes for bovine herpesvirus type 1 (BHV-1) were used to detect viral nucleic acids in infected cell cultures and clinical specimens by in situ hybridization. Hybridization signal was detected 2 hours after inoculation in the cytoplasm of infected cells, presumably representing input virus. Hybridization was first detected in the nucleus at 4 hours after inoculation; by 10 hours after inoculation, hybridization signal was detected in both nucleus and cytoplasm of almost 50% of the cells. By 15 hours after inoculation, 95% of the cells were positive. Treatment of specimens with ribonuclease or deoxyribonuclease before hybridization allowed clear differentiation of virus-specified DNA and RNA within infected cells. The BHV-1 nucleic acid sequences were detected in nasal epithelial cells obtained from inoculated calves. Since in situ hybridization provides a rapid technique for the detection of BHV-1-specified nucleic acid sequences, it should facilitate studies on the replication, pathogenesis, and diagnosis of BHV-1 infections.

Animals↗
PubMed1989

Cytomegalovirus detection by biotinylated DNA probes.

Clinical specimens from 317 patients suspected of cytomegalovirus infection were examined by immunofluorescence (IF) using monoclonal antibodies and by a biotinylated DNA probe kit after cell culture isolation. Of the 317 samples, 68 were positive by culture isolation. Of these 67 were IF positive when the cytopathic effect (CPE) was 1+ or less, whereas 56 gave positive results with DNA probes when the CPE was 2+. A further 83 specimens were examined directly by immunoperoxidase histopathology (IHP), IF and the DNA probe kit: 26 of these were positive by IHP examination, 25 by IF and only 6 by DNA probes. The sensitivity of the DNA probe kit was not satisfactory when the clinical tissue specimens were directly examined. However, the sensitivity improved considerably to 82% if the specimens were propagated first in cell culture. The IF method detected the virus before and after cell culture isolation equally well (96%-98.5%). Compared to the IF method, the DNA probe kit is costly and requires more labor and time.

Antibodies, Monoclonal↗
PubMed1997

Modulation of RNase H activity by modified DNA probes: major groove vs minor groove effects.

We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing pendant bipyridine and terpyridine groups. The ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described. DNA/RNA duplexes were formed using DNA probes designed to deliver metal complexes via either the major groove or the minor groove of the duplex. The duplexes were treated with Escherichia coli RNase H. Modifications in the major groove produced the same RNA cleavage pattern as unmodified DNA probes. However, minor groove substituents inhibited RNA cleavage over a four-base region. Comparison was made with a DNA probe containing a 2'-OMe modification. Our results support enzyme binding in the minor groove of a DNA/RNA duplex. We do not observe cleavage directly across from the modified nucleoside. The RNA cleavage efficiency effected by RNase H and a DNA probe decreases as follows: unmodified DNA > or = C-5 modified DNA >> c2'-modified DNA > C1'-modified DNA. Results with 28-mer RNA substrates roughly parallel those obtained with a 159-mer RNA target. The differences observed between low and high MW RNA substrates can be explained by a much higher enzyme-substrate binding constant for the high MW target.

Base Sequence↗
PubMed1994

Field evaluation of simplified radioactive and nonradioactive DNA probe methods for the diagnosis of falciparum malaria.

Specific DNA probe hybridization technique is one method of choice for detection of malaria infection. It provides an obvious advantage over conventional microscopy when large numbers of samples are simultaneously monitored. The method was simplified so that preparation and processing of blood specimens were all performed on membrane filters. Background signals generated from blood components were removed by treating samples spotted on the membrane with a series of buffer washes without the necessity of a protease digestion step. Hybridization was monitored using either 32P-or digoxigenin-labeled DNA probe. 849 field samples collected from various malaria endemic areas in Thailand have been evaluated by this protocol and compared with microscopic examination. Sensitivity obtained by this procedure was comparable to that of microscopy at a malaria clinic. The specificities of both types of DNA probes were better than 93%, but digoxigenin-labeled probe performed better than 32P-labeled one when the numbers of parasites were less than 25 per 200 white blood cells.

DNA Probes↗
PubMed1986

Prenatal diagnosis of autosomal dominant polycystic kidney disease with a DNA probe.

A highly polymorphic DNA probe genetically linked to the locus of autosomal dominant polycystic kidney disease was used in linkage studies for prenatal diagnosis in a nine-week fetus at risk for the disease. The fetus was judged to have inherited the polycystic kidney disease mutation, and this was confirmed by microscopic examination of the fetal kidneys at necropsy.

Abortion, Therapeutic↗
PubMed2002

Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes.

RecA protein-coated single-stranded DNA probes, known as RecA nucleoprotein filaments, bind specifically to homologous DNA sequences within double-stranded DNA targets, forming multistranded probe-target DNA hybrids. This DNA hybridization reaction can be used for sequence-specific gene capture, gene modification, and gene regulation. Thus, factors that enhance the efficiency of the hybridization reaction are of significant practical importance. We show here that the hybridization of a peptide nucleic acid (PNA) within or adjacent to the probe-target homology region significantly enhances the yield of hybrid DNA formed in the reaction between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded (css)DNA probes. The possible mechanisms and the advantages of using RecA nucleoprotein filaments in combination with PNA for genomic DNA cloning and mutagenesis are presented.

Base Sequence↗
PubMed1992

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.

Chromosomes, Human, Pair 1↗
PubMed1988

Detection of herpes simplex virus by biotinylated DNA probes.

Clinical specimens from 159 patients suspected with herpes simplex virus (HSV) were examined by monoclonal antibody immunofluorescence (IF) and by a commercial biotinylated DNA probe kit following cell culture isolation. Herpes simplex virus was isolated from 57 samples. All cultures were positive by IF when the cytopathic effect (CPE) was less than 1+ but only 49 (86%) yielded positive reaction with the DNA probe when CPE was at least 1+. A total of 54 clinical specimens was also examined directly by immunoperoxidase histopathology (IHP), IF, and DNA hybridization. Of these, 16 were positive by IHP, 15 by IF, and only five by DNA probe. The DNA probe kit was found to be reasonably sensitive only after cell culture isolation of HSV. Compared to the IF procedure, the DNA probe kit was found to be costly, labor intensive, and time consuming.

Animals↗
PubMed1990

DNA probes for the identification of Nocardia asteroides.

DNA probes for the rapid identification of Nocardia asteroides were obtained by constructing a genomic library of strain GUH-2 in the lambda cloning vector EMBL3. Of 50 recombinant clones tested, 2 were identified that hybridized with 31% of the N. asteroides strains in a reference collection without cross-hybridization with related members of the Actinomycetales. Additional libraries were then generated from selected strains of N. asteroides that had failed to hybridize with any of the GUH-2 clones. Four additional clones were obtained from these strains which, when pooled, provided DNA probes specific for all of the N. asteroides strains tested.

DNA Probes↗
PubMed1989

Use of a Chlamydia trachomatis DNA probe for detection of ocular chlamydiae.

We examined the efficacy of a Chlamydia trachomatis DNA probe in detecting ocular chlamydiae by comparing it with tissue culture isolation, direct fluorescent-antibody cytology, and clinical eye exams. In a trachoma-endemic area of Nepal, 430 Nepalese villagers were examined according to the World Health Organization trachoma grading scale. Upper tarsal conjunctival specimens from each subject were obtained for DNA probing, tissue culture, and fluorescent-antibody screening. Moderate to severe intensity of inflammation was found in 85 (21%) of 430 people studied. An additional 25 (7.2%) of 345 people with low or no intensity of inflammation also had microbiologically proven infection, which may reflect asymptomatic carriage. Compared with culture, the DNA probe had a sensitivity of 86.9% and a specificity of 91%. For direct fluorescent antibody versus culture, the values were 47.8 and 96.9%, respectively. Results from this study indicate that the DNA probe for C. trachomatis might be considered a valuable epidemiologic tool in screening trachoma-endemic populations for ocular chlamydiae.

Adolescent↗
PubMed1990

DNA probe confirmatory test for Neisseria gonorrhoeae.

A DNA probe test for the culture confirmation of Neisseria gonorrhoeae in clinical isolates was evaluated with 156 isolates of N. gonorrhoeae and 120 isolates of nongonococcal Neisseria species, organisms representative of other genera within the family Neisseriaceae, and organisms isolated on media selective for N. gonorrhoeae. The 30-min test used a chemiluminescent DNA probe that was homologous to rRNA sequences of N. gonorrhoeae. We report here a specificity and a sensitivity of 100% with the 276 clinical isolates tested, including 43 gonococcal strains that had been misidentified by other methods.

Bacteriological Techniques↗
PubMed1992

[Identification of mycobacteria by the acridinium-ester labeled DNA probes for M. tuberculosis and M. avium-intracellulare complex in culture and its clinical application].

The detectability of mycobacterium in culture by non-isotopic, chemiluminescent DNA probes for Mycobacterium tuberculosis (M. tuberculosis) and M. avium-intracellulare complex (MAC) was evaluated and compared with that by 125I-labeled DNA probe for the same mycobacteria. The sensitivity and specificity of the AE-DNA probes for MAC were 97.2% and 100%, respectively, for the conventional method and were both 100% for the 125I-labeled DNA probes. The detection limits of the AE-DNA probes tests for both M. tuberculosis and MAC were 10(5)-10(6) CFU/tube which were almost the same as those of the 125I-labeled DNA probes tests. Because the procedure is simple, rapid (it can be completed within 90 min), and safe (it does not use radioisotopes), it can be easily performed in any clinical laboratory.

Acridines↗
PubMed1999

Development of a lyophilized kit formulation for labeling of DNA probes with 99mTc.

DNA fragments such as oligodeoxynucleotides (ODNs) are under investigation for a possible utilization in nuclear medicine. Until now, experiments on 99mTc-labeled ODNs in vitro or in vivo have required the application of time-consuming procedures to obtain and control the purity of the radiolabeled compound. A lyophilized labeling kit would ease and improve the reproducibility in further investigations with this class of promising biomolecules; therefore a study was initiated to evaluate the suitability of conjugates of ODNs and a bifunctional chelating agent to be part of lyophilized kit formulations. We report here the development of the first kit for one-step labeling of oligonucleotides with 99mTc. The formulation comprises 250-500 pmol S-benzoyl-mercaptoacetyldiglycine (MAG2)-ODN phosphorothioate conjugate, 5 mg potassium sodium tartrate tetrahydrate and 100 microg stannous chloride dihydrate in a lyophilized kit. Labeling yields above 90% were reproducibly achieved after addition of 0.1-1 GBq pertechnetate and subsequent heating in a boiling water bath. Once formed, the 99mTc-MAG2-ODN complexes were stable for at least 24 h. The shelf life of the kits is at least 10 weeks when stored protected from light at room temperature, but even kits stored at 40 degrees C gave labeling yields above 90% after 10 weeks.

Chromatography, High Pressure Liquid↗
PubMed1990

Molecular differentiation of the homomorphic sex chromosomes in Megaselia scalaris (Diptera) detected by random DNA probes.

Randomly cloned DNA fragments and a poly-(GATA) containing sequence were used as probes to identify sex chromosomal inheritance and to detect differences at the molecular level between the homomorphic X and Y in the phorid fly, Megaselia scalaris. Restriction fragment length differences between males and females and between two laboratory stocks of different geographic origin were used to differentiate between sex chromosomal and autosomal origin of the respective fragments. Five random probes detected X and Y chromosomal DNA loci and two others recognized autosomal DNA loci. One random probe and the poly(GATA) probe hybridized with both sex chromosomal and autosomal restriction fragments. Most of the Y chromosomal restriction fragments were conserved in length between the two stocks while most of the X chromosomal and autosomal fragments showed length polymorphism. It was concluded, therefore, that the Y chromosome contains a conserved segment in which crossover is suppressed and restriction site differences have accumulated relative to the X. These chromosomes, therefore, conform to a theoretically expected early stage of sex chromosome evolution.

Animals↗
PubMedDate not supplied

Application of DNA probes to antimicrobial susceptibility testing of Legionella pneumophila.

The commercial availability of a DNA probe assay for the detection of Legionella (Gen-Probe Incorporated, San Diego, CA) provides a unique opportunity to investigate the application of this technology to antibiotic susceptibility testing of L. pneumophila. We examined the ability of erythromycin, rifampin, and ciprofloxacin to kill L. pneumophila in buffered ACES-yeast extract broth (YEB). The test organism was incubated for a total of 96 hr in the presence of 10 micrograms/ml erythromycin, 1 micrograms/ml rifampin, or 1 micrograms/ml ciprofloxacin. Growth was monitored at 24-hr intervals by quantitative plating and the DNA probe assay. The correlation between organism concentration [colony-forming units (CFU) per ml] and DNA probe activity (counts per min) was excellent (r = 0.97). The percent decrease in CFU/ml at 96 hr relative to control counts was greater than 99% for erythromycin, rifampin, and ciprofloxacin. The percent decrease in CPM at 96 hr versus control was 87% for erythromycin, 89% for rifampin, and 93% for ciprofloxacin. This data documents a novel application of DNA probe technology, which may be useful in future studies of in vitro susceptibility of Legionella to various agents.

Anti-Bacterial Agents↗