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Query: UNIPROT:P06889 (Mol)
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In situ hybridization (ISH) provides a means for identifying viral genomes in the context of tissue pathology. We have developed a specific and sensitive ISH probe for the detection of cytomegalovirus (CMV) DNA in formalin-fixed, paraffin-embedded tissue sections. Digoxigenin-11-dUTP was incorporated into a 435-base pair fragment of the CMV Major Immediate Early (MIE) gene with use of the polymerase chain reaction (PCR). Hybridized probe was detected by reaction with antidigoxigenin antibody coupled to alkaline phosphatase and chromogenic substrates. This method has detected CMV infection in routine clinical specimens from a variety of tissue types, including colon, kidney, liver, and stomach. Infection in cells with and without characteristic inclusions is revealed with this probe. The background is so low that single infected cells are detected unambiguously. No cross-hybridization was observed with cells infected with other viruses of Herpesviridae. This approach may be useful for producing probes for the detection of other viral genomes in tissue sections.
Diagn Mol Pathol 1994 Sep
PMID:PCR production of a digoxigenin-labeled probe for the detection of human cytomegalovirus in tissue sections. 798 96

A capture hybridization technique in microplate has been developed for the identification of polymerase chain reaction (PCR) amplified B19 DNA fragment in clinical specimens. The amplified 104 bp B19 DNA fragment, located in the gene coding for structural proteins, was directly labelled during the amplification reaction by incorporation of digoxigenin-labelled dUTP. The amplified product was then captured by a probe immobilized on microplate wells. The capture hybridization reaction was visualized as an enzyme-linked immunosorbent assay using anti-digoxigenin Fab fragment labelled with peroxidase. Thirty-five serum samples were tested by our capture hybridization assay and the results were in accordance with the results obtained by Southern blot analysis of PCR amplified product. Our microplate capture hybridization assay showed a high sensitivity and reproducibility and appears to be a practical and reliable test for routine screening of B19 parvovirus DNA in clinical specimens.
Mol Cell Probes 1993 Dec
PMID:Microplate capture hybridization of amplified parvovirus B19 DNA fragment labelled with digoxigenin. 814 76

A highly sensitive in situ hybridization procedure was established using digoxigenin-11-dUTP-labeled probes that were prepared by the polymerase chain reaction (PCR). By using 12 sets of primers, BamHI-W fragment of the Epstein-Barr Virus (EBV) was amplified with labeled substrate in individual PCRs. Then the 12 probes (average size, 120 base pairs) were mixed and hybridized with cultured and RNase-treated Namalwa cells, which contain two copies of EBV genomes per cell. The strength of the signals was much stronger as compared with random-primed probe. Our results indicate that the size-averaged PCR probes magnified the sensitivity for detecting low copy numbers of virus genomes by in situ hybridization and that this technique has the potential for investigating latent virus infection in other clinical situations.
Diagn Mol Pathol 1993 Jun
PMID:Detection of low copy numbers of Epstein-Barr virus by in situ hybridization using nonradioisotopic probes prepared by the polymerase chain reaction. 826 74

Invertebrates that contain endosymbiotic chemoautotrophic eubacteria are widely distributed in a variety of reducing marine habitats, including deepsea hydrothermal vents. The mechanisms of symbiont transmission in these invertebrates are not understood. To test the hypothesis that symbionts are transmitted via the eggs of hosts, we used group-specific hybridization probes complementary to 16S ribosomal RNAs (rRNAs) to look for symbionts in eggs and ovaries. 16S rRNA sequences were examined for domains unique to the symbionts of three vent animals: Calyptogena magnifica, Bathymodiolus thermophilus, and Riftia pachyptila. Three 16S rRNA-directed oligodeoxynucleotide hybridization probes (CG-1255R, RP-1243R, BT-1255R) specific for these endosymbionts were synthesized and evaluated by dot-blot hybridization. At higher stringencies, all three probes showed a high degree of specificity for their target endosymbionts rRNAs. The probes were also used as polymerase chain reaction (PCR) primers for detection of the symbiont 16S rRNA genes in genomic DNA isolated from host tissues known to contain symbionts. All three symbiont-specific probes were highly sensitive and specific as PCR primers; they successfully amplified 1 pg target DNA. However, all amplifications of extracted egg DNA from the vestimentiferan R. pachyptila with either universal eubacterial (Eub A/B) or the Riftia symbiont-specific (RP-1243R/Eub B) primer sets were unsuccessful. Nonradioactive in situ hybridizations were performed on ovarian tissue from the vestimentiferan Ridgea piscesae using RP-1243R, 3' end-labeled with digoxigenin-11-dUTP (Boehringer Mannheim). The probe was subsequently detected with an alkaline phosphatase-conjugated immunoglobulin G antibody specific for the digoxigenin moeity. The probe bound only to the tissue of R. pisceasae coincident with the known location of symbiont cells and was not detected in any region of the ovary. These data indicate that transovarial symbiont transmission in the vestimentiferans does not take place and that symbiont acquisition is probably a post-spawning event.
Mol Mar Biol Biotechnol 1993 Feb
PMID:Identification and localization of bacterial endosymbionts in hydrothermal vent taxa with symbiont-specific polymerase chain reaction amplification and in situ hybridization techniques. 836 89

A modified reverse dot-blot assay was developed and used in combination with a nested PCR amplification of hly A for the specific detection of Listeria monocytogenes. The deoxynucleotides and digoxygenin-11-dUTP concentrations in the PCR were optimized for maximal sensitivity and economy. For the dot-blot hybridization, digoxygenin-labelled PCR products were directly spotted on nylon membrane-bound poly-dT tailed capture probes and the signal detection was either colorimetric or chemiluminescent. With crude cell lysates, the total assay could be completed in about 6 h with a detection limit between 2 and 25 colony forming units (cfu) per PCR. The assay was then tested for its applicability to environmental sampling of artificially contaminated aluminum surfaces. For environmental sampling, the assay requires an additional overnight enrichment step and has a sensitivity corresponding to 5 cfu per 25 cm2 of inoculated surface.
Mol Cell Probes 1993 Jun
PMID:A combined modified reverse dot-blot and nested PCR assay for the specific non-radioactive detection of Listeria monocytogenes. 836 65

A one-step polymerase chain reaction (PCR) was used to synthesize a digoxigenin-labelled probe, 176 bp long, for the detection of human polyomavirus BK (BKV). A 104 bp-long digoxigenin-labelled probe was generated by 'nested' PCR for the detection of human parvovirus B19 (virus B19). In both cases the whole viral genome was used as template. Different amounts of template as well as different percentages of dTTP substituted by digoxigenin-dUTP (dig-dUTP) in the reaction mixture were employed in order to determine the optimum conditions for the labelled probe synthesis. The sensitivity and the specificity of these PCR-produced probes, together with the simplicity and the reduced time scale of the procedure, suggest the potential of this technique as an additional method for preparing non-radioactive molecular probes for routine diagnosis of viral infections.
Mol Cell Probes 1993 Feb
PMID:Polymerase chain reaction for synthesis of digoxigenin-labelled DNA probe: application to parvovirus B19 and to polyomavirus BK. 838 14

A colorimetric assay for the detection of PCR-products is described. The assay is based on amplification of DNA in the presence of digoxigenin-dUTP. After immobilization of the PCR products to a microtitre plate, amplified DNA could be detected colorimetrically. The sensitivity of this colorimetric assay was equal to gel-analysis allowing the detection of 100 fg of template DNA. Here, we show that it can be used to detect Mycobacterium leprae DNA in biopsy specimens from leprosy patients. The simplicity and the low degree of variation make this assay an alternative to gel-analysis.
Mol Cell Probes 1993 Feb
PMID:A simple colorimetric assay for detection of amplified Mycobacterium leprae DNA. 845 42

To characterize the process by which the mammalian nucleotide excision repair complex interacts with DNA to recognize and repair lesions, we have investigated the size and distribution of repair patches induced by human cell extracts in ultraviolet light-irradiated plasmid DNA. Repair synthesis was carried out in a buffer substituting biotinylated dUTP for dTTP, to allow repair patches to be detected by electron microscopy after streptavidin/colloidal gold labelling. Individual repair events on circular plasmids that had undergone repair synthesis in cell extracts were scored as gold particles bound specifically to irradiated molecules. Samples of over 2000 irradiated and unirradiated plasmids were counted. Repair synthesis at ultraviolet light photoproducts typically replaced about 30 nucleotides, since 69% of patches contained only one particle of 10 nm gold and 24% of patches contained two gold particles (each covering approx. 29 nucleotides). In addition, the ordering of repair events among damaged plasmids closely fitted a Poisson distribution, indicating that repair of lesions is achieved via a non-processive, random diffusion mechanism. This suggests that the repair complex is not intrinsically processive.
J Mol Biol 1993 May 20
PMID:Electron microscopy of DNA excision repair patches produced by human cell extracts. 851 Jan 46

A quantitative method of polymerase chain reaction (PCR) using both digoxigenin and radioactive labelled probes has been used for the detection of the c-erbB-2 proto-oncogene amplification in breast carcinomas with formalin-fixed paraffin-embedded tissue sections. c-erbB-2 proto-oncogene amplification has been demonstrated in 14 infiltrating ductal carcinomas. The technique consisted of the co-amplification of c-erbB-2 and IFN-gamma (interferon-gamma) genes. The latter was considered as a single copy gene per genome-equivalent. The aim of this study was to compare two quantitative PCR techniques based on the incorporation of either digoxigenin-11-dUTP or 32P-dCTP, during amplification. For the colorigenic method, using the Dig system, after electrophoresis and transfer, the specific bands were revealed with a chromogenic substrate of phosphatase. Their intensity estimated by scanning photometry following blot transparisation. After electrophoresis, the radioactive gel was submitted to radioautography and the band intensities evaluated by scanning spectrophotometry. For the 14 samples, a good agreement between both methods was noted. The colorigenic method is a valuable alternative to radiolabelling due to: i) time saving, ii) reagent conservation, iii) safe manipulation and iv) sensitivity of the same order for both methods.
Cell Mol Biol (Noisy-le-grand) 1995 Nov
PMID:Determination of amplification level of the c-erbB-2 proto-oncogene in human breast carcinomas: a comparative study between non-radioactive and radioactive labelling. 859 75

PCR product sizing on ethidium bromide-stained gels, coupled with Southern transfer and hybridization with nonisotopic probes, is an effective way of detecting t(14;18)(q32;q21). We evaluated an alternative ELISA-based test for detecting amplified t(14;18) products. Digoxigenin (DIG)-labeled dUTP is incorporated in a standard PCR method for amplification of bcl-2 major breakpoint region (mbr) rearrangements. The product is hybridized to a specific biotinylated DNA probe internal to the mbr primer, placed in streptavidin-coated wells of a microtiter plate, and detected with a alkaline phosphatase-conjugated anti-DIG antibody and enzyme substrate (pNpp). The colorimetric product is quantitated by an automated optical density (O.D.) reader. We evaluated 13 mbr-positive follicular lymphomas (FL), five mbr-negative B-cell neoplasms (BCN), 16 reactive lymphoid hyperplasias (RLH), 14 cases of Hodgkin's disease (HD), and normal peripheral blood samples from 20 healthy volunteers. All samples were evaluated in duplicate on separate plates. Positive [t(14;18)-containing cell line] and negative [cell line without t(14;18); master mix only] controls, and a standard curve were included with each run. Numerical O.D. readings from the specific hybridization assays revealed differences between FL and the other categories. All FL had an O.D. reading at > 2.0. The vast majority of RLH, HD, BCN, and normal peripheral blood samples showed O.D. readings well below 2.0. Specifically, 13/16 RLH and all HD, BCN, and normal peripheral blood samples had an O.D. of < or = 0.6 in all runs. The three outliers, which were all < 2.0, may represent the low level detection of t(14;18)-containing cells in RLH similar to previous reports. Moreover, all but four RLH had O.D. readings above the background negative controls, suggesting that rare t(14;18)-containing cells may have been present in these samples, as well. Dilution studies estimate that this assay is capable of detecting 1 t(14;18)-containing cell in approximately 10(5) cells, a greater level of sensitivity than can be obtained with gel visualization alone. We conclude that this semi-automated, potentially quantifiable ELISA-based system is a useful, objective and reproducible alternative hybridization procedure for verifying PCR product specificity in this setting.
Diagn Mol Pathol 1996 Jun
PMID:Semi-automated ELISA-based detection system for verifying the authenticity of amplified t(14;18)-containing products. 872 98


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