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PubMed1990

[Diagnostic nucleic acid hybridizations for infectious diseases].

The use of nucleic acid hybridization techniques has expanded into many areas, including studies of gene structure and function, routine diagnosis of human, animal and plant diseases, and also forensic science. In situ hybridization is one of the techniques currently available for nucleic acid hybridization and has some distinct advantages compared with standard techniques such as dot-blot, Southern or Northern hybridization, in which the histological structure is lost during extraction of nucleic acids. On the other hand, immunohistochemical staining is one branch of histochemistry that has received considerable attention in recent years as a very sensitive method for localization of specific proteins and other antigenic macromolecules within tissues and cells. This technique has also been widely used for clinical diagnosis and in various fields of research in medical science and biology. Automation of colorimetric in situ hybridization and immunohistochemistry would greatly contribute to the ease of introducing these techniques for routine pathological diagnosis and would improve the reproducibility of the assay. In this review, author will describe the development of an automated method for in situ hybridization and immunohistochemical staining using an automatic machine for both procedures.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

[Demonstration of the tick-borne encephalitis virus in ticks by means of nucleic acid hybridization].

The method of molecular hybridization of nucleic acids (MHNA) is compared to the traditional bioprobe technique in the study of virus carriership of I. persulcatus ticks collected in the South and in the North of the area of coniferous and broad-leaved forests in the Khabarovsk Territory. Higher sensitivity of the MHNA method than that of the bioprobe was demonstrated by studying the tick pools: 4.9% versus 1.6% (1985), 18.2% versus 3.6% (1986). Virus carriership studied by MHNA in isolated ticks comprised 8.6% (1985) and 10.8% (1986) in the south of the region and 20.0% (1985)--in the north; average content of RNA of the tick-borne encephalitis virus comprised 10 pg. MHNA allows massive studies of individual ticks and better indication of the virus to be carried out; the response can be obtained in 24-48 hours.

Animals↗
PubMed1987

Detection of dengue virus RNA using nucleic acid hybridization.

Conditions for using slot-blot nucleic acid hybridization to quantitatively detect dengue-2 virus using a radiolabelled cDNA probe, pVV17, were determined. As little as 11 plaque-forming units of virus were detected using a hybridization mixture without formamide and performing the test at 70 degrees C. While predominantly serotype-specific using stringent (65 degrees C) washing conditions, the probe detected all four dengue virus serotypes using astringent (28 degrees C) washing conditions. No significant qualitative differences were detected using Thai dengue-2 viruses isolated over a 10-year period. High titered, anti-flavivirus antibodies blocked virus detection by an antigen capture, enzyme-linked, immunosorbent assay or by intrathoracic inoculation of Toxorhyncites mosquitoes, but not by nucleic acid hybridization. The appearance of virus-specified RNA coincided with the detection of antigen in infected C6/36 (Aedes albopictus) cells by immunofluorescence, or in cell culture supernatants by the antigen capture method. The method has potential as a diagnostic tool for identifying dengue viruses in clinical and field specimens.

Animals↗
PubMed1989

[Probe seeks gene: nucleic acid hybridization as a current contribution to virologic diagnosis].

Molecular nucleic acid hybridization is based on the ability of single-stranded DNA/RNA to form hybrids with complementary labeled nucleic acids. This review shortly describes the components of this technique and presents the most important hybridization methods (spot-, Southern blot-, in situ-hybridization). The (potential) applications of nucleic acid hybridization as a diagnostic tool are discussed (e.g., for viruses which grow insufficiently or not at all in cell culture; virus latency; viruses with labile or antigenically variable envelope proteins, resp.; for virus classification) and yet existing limitations are indicated. An impetus for this technique in means of diagnostic application is expected to result from the Polymerase Chain Reaction (PCR) in the next future.

Animals↗
PubMed1989

An assay for quantitative nucleic acid hybridization on membrane filters.

A quantitative nucleic acid hybridization assay based on a 6-mm-diameter nitrocellulose membrane filter and only 20 microliters of hybridization mixture per determination is described. As a consequence of the small ratio of hybridization volume to membrane surface, the hybridization rates reached in this system are higher than those obtained at volume to surface ratios of conventional protocols, allowing even RNA at very low concentrations to complete hybridization. This advantage, together with the high reproducibility and hybrid stability obtained with the assay, increases the ability of the filter hybridization technique to analyze quantitatively minute amounts of RNA.

Bacteriophages↗
PubMed1987

A homogeneous nucleic acid hybridization assay based on strand displacement.

A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement.

Base Sequence↗
PubMed1994

Identification of Bacillus strains isolated from milk and cream with classical and nucleic acid hybridization methods.

A total of 529 bacterial strains have been isolated from milk and cream sampled at different sites in a dairy production plant under conditions selective for aerobic sporeforming bacteria. Identification with classical methods based on morphological, physiological and biochemical criteria showed Bacillus licheniformis to be the most frequently occurring Bacillus sp. The investigation also revealed 62 unidentified strains. Classical identification methods were time consuming (3-7 d), lacked specificity and--because of their dependence on phenotypic gene expression--sometimes produced ambiguous results. Consequently, a colony hybridization method developed for the identification of B. licheniformis strains and using nonradioactive labelled 23S rRNA targeted oligonucleotide probes was also used. Identification of B. licheniformis with both classical and hybridization methods revealed diverging identification results for 70 strains.

Animals↗
PubMed1987

[Technic of nucleic acid hybridization and its significance for diagnostic problems].

Nucleic acid hybridization techniques (e.g., Southern blotting and in situ hybridization) gain increasing importance in laboratory medicine, as demonstrated by examples in this paper. The molecular basis of this approach to detect nucleic acids homologous to known DNA probes is explained. It is concluded that this technique has advantages in the detection of pathogens compared to other methods used in laboratory medicine.

Clinical Laboratory Techniques↗
PubMed2005

Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

BACKGROUND: A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. METHODS: The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. CONCLUSIONS: The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

Animals↗
PubMed2001

Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes.

Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2-4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68 degrees C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single-copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.

Animals↗
PubMed2004

A robust technique for assembly of nucleic acid hybridization chips based on electrochemically templated chitosan.

A nucleic acid hybridization assay was assembled onto a robust and readily addressable silicon-based chip using polysaccharide chitosan as a scaffold for the covalent coupling of probe DNA to the chip's surface. Chitosan is a unique polymer, ideally suited for this application because its net charge and solubility are pH dependent. Specifically in this work, gold-patterned electrodes were created using standard photolithographic techniques, chitosan was electrodeposited in a spatially resolved manner onto the polarized electrodes, probe DNA was covalently assembled onto the chitosan, and both DNA:DNA and DNA:mRNA hybridization detection schemes were evaluated. Hybridization of target nucleic acid was quantifiable, reproducible, and robust; the surface was regenerated and rehybridized up to eight times without loss of signal. Finally, transcriptional upregulation of the Escherichia coli chaperone, DnaK, which is an indicator of cellular stress, was observed using the hybridization chip sandwich assay. Thus, this method enables rapid and facile monitoring of gene expression in a format that is reusable and requires minimal reagent quantities.

Chitin↗
PubMedDate not supplied

Comparative studies of nucleic acid hybridization assay for Listeria in foods.

A nucleic acid hybridization assay has been developed for Listeria spp. in dairy foods and environmental samples. The assay is based on detection of unique Listeria 16S rRNA sequences by using a 32P-labeled synthetic DNA probe. Inclusivity and exclusivity of the probe were confirmed with 139 Listeria isolates representing all known species, and 73 non-Listeria bacterial strains. In this paper, we present results from our preliminary studies comparing the hybridization assay with conventional culture on a total of 575 specimens that represent a variety of inoculated and uninoculated foods and environmental samples. The assay, which is done in a filter manifold format after 2 days of cultural enrichment, requires a total assay time of less than 2.5 days. The false-negative rate for all sample groups tested using the GENE-TRAK hybridization assay was less than the rate for culture. Thus, the new assay allows rapid screening of the indicated product groups and provides reliable numerical results.

Dairy Products↗
PubMed1997

Optimal configuration of pyrene excimer induced by the excimer-forming two-probe nucleic acid hybridization method.

Excimer-forming two-probe nucleic acid hybridization (ETPH) method with pyrene as a fluorophore enables homogeneous hybridization assays. We examined the effect of linker length between a pyrene residue and a terminal sugar moiety on Tm of hybrids in the presence of 20% dimethylformamide (DMF). The results including those of CD measurements indicated no interaction of pyrene residues with the duplex formed between a target 32-mer and a pyrenemethyliodoacetamide-introduced 16-mer probe (PMIA-P5)/a pyrenebutanoic acid-introduced 16-mer probe (PBuA-P3), which is the best pair of probes for intense excimer emission. This was also supported by a computer-assisted molecular modeling using Insight II and Discover software.

DNA↗
PubMedDate not supplied

[Use of a method of molecular nucleic acid hybridization for the rapid diagnosis of influenza].

A highly sensitive method of pinpoint hybridization of nucleic acids on nitrocellulose filters using 32P-labeled pHA plasmid carrying a DNA copy of hemagglutinin gene of influenza A/Udorn/307/72 (H3N2) was developed which permitted specific detection of minimal amounts of RNA (units of pikograms) of influenza A virus with H3 serotype hemagglutinin. The method of pinpoint hybridization was used for the detection of RNA of influenza A (H3 serotype) in nasopharyngeal washings of patients with acute respiratory diseases during the influenza outbreak of February-March, 1984. In parallel, the presence of viral antigen was determined by direct immunofluorescence using H3N2 antiserum, and the diagnosis of influenza was confirmed by the clinical picture of the disease. The results indicate that the pinpoint hybridization method may be used for rapid diagnosis of influenza as a highly sensitive and specific tool.

Adolescent↗