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PubMed1998

Electron microscopic in situ hybridization of digoxigenin-dUTP-labelled DNA probes with Drosophila melanogaster polytene chromosomes.

We report a simplified method of electron microscopic (EM) in situ hybridization for standard squashes of Drosophila melanogaster polytene chromosomes using digoxigenin-11-dUTP labelled DNA probes. The method is efficient and reproducible: its high resolution and specificity were demonstrated for the transformed strain 148, in which the insertion was localized precisely as a new thin band both by conventional EM and according to our method. In addition, the method was applied to the fine mapping of the developmentally regulated gene muscle-blind (mbl). On the one hand, mbl was shown to cover the 54B1-2 large band and the adjacent interbands in the 2R polytene chromosome. On the other hand, the use of distantly located DNA probes in the mbl gene allowed us to orientate the transcription unit in the chromosome.

Animals↗
PubMed1993

Localization of xanthine dehydrogenase mRNA in horse skeletal muscle by in situ hybridization with digoxigenin-labelled probe.

In situ hybridization was used to localize xanthine dehydrogenase (XDH) mRNA in horse skeletal muscle. Capillary endothelial cells were found to express XDH, but muscle cells did not give any signal. The digoxigenin-labelled probe was produced by PCR with primers based on the cDNA sequence of mouse XDH and horse lung cDNAs. A 4.3 kb mRNA was detected in a Northern blot.

Animals↗
PubMed1998

Detection of pathogenic Yersinia enterocolitica using a digoxigenin labelled probe targeting the yst gene.

A 145 base pair digoxigenin-d-UTP-labelled probe, specific for pathogenic Yersinia enterocolitica heat-stable enterotoxin yst gene, was prepared by PCR. The probe was used in DNA-DNA colony hybridization and dot-blot hybridization assays. The specificity of the probe was confirmed using 52 strains representing all Yersinia spp., except Y. pestis. Out of a total of 25 Y. enterocolitica strains screened, the probe correctly identified all 18 pathogenic strains. Among the other Yersinia spp. screened, only one strain of Y. kristensenii was positively detected by the yst probe but could be differentiated by its weak signal response as compared with that obtained by pathogenic strains of Y. enterocolitica.

Bacterial Toxins↗
PubMed1999

Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus.

The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the use of a thermolabile haemolysin (tlh) gene probe is proposed. An alkaline phosphatase (AP)-labelled probe was evaluated for specificity against 26 strains of V. parahaemolyticus, 88 strains of other Vibrio species and 10 strains of non-vibrio species. Of the 124 isolates tested, the probe hybridized only with the 26 strains of V. parahaemolyticus, indicating species specificity. Two hundred and six suspect V. parahaemolyticus isolates from oysters were tested by this probe and API-20E diagnostic strips; there was 97% agreement between results. A digoxigenin (DIG)-labelled probe for detection of the tlh gene fragment was prepared by PCR and compared with the AP-labelled probe. When tested on 584 suspect V. parahaemolyticus isolates, results obtained with the AP- and DIG-labelled probes were in 98% agreement. These results suggest that the probes are equivalent for detection of the V. parahaemolyticus tlh gene.

Alkaline Phosphatase↗
PubMed2003

Digoxigenin-labelled peptide nucleic acid to detect lactobacilli PCR amplicons immobilized on membranes from denaturing gradient gel electrophoresis.

AIMS: To develop a digoxigenin (DIG)-labeled peptide nucleic acid (PNA) probe for the detection of Lactobacillus-related genera amongst eubacterial amplicons obtained from vaginal samples using denaturing gradient gel electrophoresis (DGGE) blots. METHODS AND RESULTS: Part of the 16S rRNA gene sequence was used as a target for the PNA probe. After confirming probe specificity using chromosomal DNA from species and isolates that have been detected in the urogenital tract, it was successfully used to detect lactobacilli amplicons generated using eubacterial-specific 16S rRNA gene-targeted primers from vaginal tract samples immobilized on membranes from DGGE. CONCLUSIONS: The Lactobacillus-specific PNA probe could distinguish between DNA fragments from lactobacilli in a DGGE gel from other bacterial species, including those that migrated to a similar position. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the DIG-labelled PNA probe on blots of eubacterial PCR products from DGGE gels can be used to specifically detect lactobacilli in complex vaginal samples.

DNA, Bacterial↗
PubMed1992

Central venous catheter sepsis caused by unusual Gordona (Rhodococcus species: identification with a digoxigenin-labeled rDNA probe.

We describe central line sepsis caused by Gordona (Rhodococcus) species in two patients, which complicated receipt of long-term total parenteral nutrition at home. Species identification was attempted by conventional biochemical analysis and analysis of polymorphisms in the ribosomal RNA genes with use of a digoxigenin-labeled rDNA probe. Using these techniques, we identified our first patient's isolate as Gordona terrae. The isolate from our second patient was biochemically atypical and could not be reliably matched to any of the recognized Gordona (Rhodococcus) species. To our knowledge, these patients are the first to have been reported with systemic infection caused by Gordona (Rhodococcus) species. The first patient's infection resolved after 6 weeks of intravenous therapy with vancomycin with the catheter left in situ; however, infection in the second patient required catheter removal for cure. These cases show that immunocompetent patients receiving long-term parenteral nutrition may develop central line infections with these unusual species of microorganisms.

Actinomycetales Infections↗
PubMed1992

Identification and epidemiological typing of clinical and environmental isolates of the genus Rhodococcus with use of a digoxigenin-labeled rDNA gene probe.

Rhodococcus species are ubiquitous in the environment, and several species have been reported to have pathogenic potential for humans. Rhodococcus equi, in particular, has been reported to cause infections in patients with AIDS. However, the identification of Rhodococcus species with use of conventional biochemical tests is problematic, and no simple and reproducible method exists for their rapid identification and differentiation. We found that the type strains of the 20 recognized species in the genus Rhodococcus could be clearly distinguished with use of a combination of the Pvu II and Pst I rRNA gene restriction endonuclease patterns and a digoxigenin-labeled Escherichia coli rDNA probe. Analysis of four clinical or environmental isolates confirmed as Rhodococcus bronchialis showed no interstrain variation of rRNA gene bands. Analysis of 15 isolates confirmed as R. equi from 13 patients showed 11 different rRNA gene patterns. No discernible difference was observed in the ribotype patterns between R. equi isolates from patients for whom AIDS had been diagnosed and those from patients who did not have AIDS, and there was no evidence of geographic clustering of R. equi ribotype patterns. Three of five Rhodococcus species isolates that could not be differentiated with use of conventional biochemical methods were identified with use of ribotype analysis. Therefore, ribotype analysis may provide an important adjunct to current biochemical identification of environmental and clinical isolates of Rhodococcus species.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Use of digoxigenin-labelled ampicillin in the identification of penicillin-binding proteins in Helicobacter pylori.

Amoxycillin is used in current therapeutic regimens to treat the infection caused by the human gastric pathogen, Helicobacter pylori. The penicillin-binding proteins (PBPs) are the primary targets for the beta-lactam antibiotics, such as amoxycillin, and are involved in the terminal stages of peptidoglycan synthesis. They also play active roles in the determination and maintenance of cellular morphology. It was believed that an organism with a complex morphology, such as H. pylori, would have more than the three PBPs previously suggested. Using digoxigenin-labelled ampicillin (DIG-ampicillin), we report the identification of eight PBPs in H. pylori with masses of 72, 62, 54, 50, 44, 33.5, 30.5 and 28 kDa. A smaller (21 kDa) ninth band was also detected, which may represent another PBP. However, the relatively small size of this apparent PBP raises questions as to whether this is a true PBP. In an attempt to identify the PBPs to which amoxycillin preferentially binds, amoxycillin was used in competition assays with DIG-ampicillin. It appeared that amoxycillin inhibited the binding of DIG-ampicillin to only the 72 kDa PBP. The experimental data were also compared with the seven putative PBPs identified in the two published H. pylori genomes, most of which correlate with the experimental data. To investigate further the properties of these PBPs, the seven putative PBP genes identified in the H. pylori genomes were examined. The derived amino acid sequences of the putative PBPs were examined for the three characteristic motifs found in all conventional PBPs, SXXK, SXN and KTG. We were able to determine that all of the putative PBPs had at least one of these motifs, but none possessed all three motifs with the characteristics of conventional PBPs. These findings suggest that the PBPs of H. pylori are unique.

Ampicillin↗
PubMed1994

Digoxigenin-tagged anti-GFAP and multiple labelling of human glia, vessels and beta-amyloid.

Cytochemical procedures for the combined demonstration of astroglia, microglia, blood vessels and beta-amyloid in the human brain were developed. These multiple label experiments include the first adaptation of the digoxigenin-antidigoxigenin technique to immunocytochemistry by using digoxigenylated antibodies directed against the glial fibrillary acidic protein and its visualization in astrocytes with an antidigoxigenin-peroxidase conjugate and diaminobenzidine as chromogen. Furthermore, the specific labelling of microglial cells was performed with the novel enhanced polymer one-step staining technique, non-interfering with conventional detection systems. The demonstration of spatial relationships between glial and vascular components in normal tissue and their alterations, e.g. in Alzheimer's disease, might provide insights into the time course and localization of pathological events.

Alzheimer Disease↗
PubMed1994

Growth hormone and prolactin gene expression in human densely and sparsely granulated somatotroph adenomas by in situ hybridization with digoxigenin-labeled probes.

We studied growth hormone (GH) and prolactin (PRL) messenger ribonucleic acids (mRNAs) in 14 cases with densely granulated somatotroph (DG) adenomas and 10 cases with sparsely granulated somatotroph (SG) adenomas using in situ hybridization with digoxigenin-labeled probes and correlated these data with their immunohistochemical results. A good correlation between in situ hybridization results and immunohistochemical data was found in most cases examined. The DG adenomas generally had a diffuse and intense GH immunoreactivity and GH hybridization signal, whereas in SG adenomas the number of cells exhibiting a GH mRNA signal and the strength of the GH mRNA signal in these cells were relatively lower than those of DG adenomas, indicating that lower expression of the GH mRNA signal is responsible for lower GH production in SG adenomas. In addition, PRL mRNA expression differed in the two types of adenoma; in DG adenomas, seven cases (50%) expressed PRL mRNA signal with a focal or scattered distribution despite normal serum PRL levels, but two showed no PRL immunoreactivity. In contrast, in SG adenomas, only one case contained a few cells possessing a PRL mRNA signal despite having no PRL immunoreactivity. It can be concluded that DG and SG adenomas, which have been considered variants of the same tumor, display definite differences as to GH or PRL gene expression in each type of adenoma.

Adenoma↗
PubMed1994

PCR production of a digoxigenin-labeled probe for the detection of human cytomegalovirus in tissue sections.

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.

Base Sequence↗
PubMed1991

Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides.

Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5'-end or enzymically at the 3'-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody-enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gram-negative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.

Antibodies, Bacterial↗
PubMed1994

Detection of human papillomavirus type 60 in plantar cysts and verruca plantaris by the in situ hybridization method using digoxigenin labeled probes.

BACKGROUND: Since 1987, many cases have been reported in which human papillomavirus (HPV) could be associated with epidermoid cysts of the palms and soles. In 1989, the HPV found in an induced epidermoid cyst was cloned and named HPV 60. OBJECTIVE: Our purpose was to examine the relationship of HPV 60 to plantar cysts. METHODS: Ten cases of plantar cysts and two cases of verruca plantaris containing single inclusion bodies were obtained by biopsy and examined by the in situ hybridization (ISH) method with a digoxigenin labeled HPV 60 DNA probe. RESULTS: Six of ten cases were found to have cytopathic effect (CPE) in the cyst wall and inner contents of the cyst. HPV 60 was detected in these cases by ISH. Two cases of verruca plantaris showing CPE also demonstrated the presence of HPV 60. Of the six HPV type 60 positive cases, four cases also showed CPE in the overlying epidermis. CONCLUSION: At first, punctate keratotic lesions are induced by HPV 60. HPV 60 induces irregular elongation of the rete ridges into the dermis. Plantar cysts probably form as a result of the traumatic implantation of HPV 60 infected epidermis with irregularly long and narrow rete ridges into the dermis.

Adult↗
PubMed1990

Detection of provirus in an HTLV-II producer CD8+ T cell line by polymerase chain reaction combined with digoxigenin-ELISA method.

A human T-cell leukemia virus type II (HTLV-II) producer cell line, designated HTLV-IIA, was established by cocultivation with leukocytes from an anti-human T-cell leukemia type I (HTLV-I) antibody-positive white male intravenous drug abuser and a healthy Japanese female. The cell line was examined for viral antigens by the indirect immunofluorescence method. The cytoplasm of over 80% of the cells was brilliantly stained. Cytogenetically, the cell line has a normal female karyotype. Electron microscopy of the HTLV-IIA disclosed many C-type retrovirus particles of mature, immature and non-cored types in the extracellular spaces. The surface markers of the transformed cells are CD2+, CD3+, CD4- and CD8+. To distinguish between HTLV-I and HTLV-II infection in the cell line, a method for detection of the HTLV-II provirus was developed by combining the polymerase chain reaction method with digoxigenin-enzyme-linked immunosorbent assay method.

Adult↗
PubMed1962

Chemical structure and pharmacological activity of some derivatives of digitoxigenin and digoxigenin.

A series of derivatives of digitoxigenin and digoxigenin were prepared and tested for toxicity in the cat and the guinea-pig and on the isolated heart of the 48-hr chick embryo, and for inotropic activity on the cat isolated papillary muscle and the guinea-pig Langendorff heart. The order of relative potency of the compounds remained the same whether they were tested for toxicity or for positive inotropic activity. There are three molecular centres in the cardiac aglycone that are linked closely with cardiac activity. These are: (a) an OH at carbon-3 which can be combined as a glycoside, thus enhancing activity, or esterified or oxidized, producing compounds of lower activity; the maximum intensity of the inotropic response was reduced in the less potent compounds; (b) a 14-beta-OH associated with a cis C-D ring junction, alteration of which abolished activity; (c) an unsaturated cyclobutenolide ring which cannot be reduced without a great decrease in activity.

Animals↗
PubMedDate not supplied

Non-radioactive oligotyping for HLA-DR1-DRw10 using polymerase chain reaction, digoxigenin-labelled oligonucleotides and chemiluminescence detection.

We describe a rapid non-radioactive DNA typing of the serological types DR1-DRw10 using polymerase chain reaction (PCR)-amplified DNA and 15 sequence-specific oligonucleotides (SSO) which are labelled enzymatically at their 3' end with one digoxigenin (DIG). The hybridized SSOs were detected using anti-DIG alkaline phosphatase and Fab fragments and visualization was obtained with the chemiluminescent substate 3-(2'-spiroadamantan)-4-(3''-phosphoryloxy)-phenyl-1,2-di o xetan (AMPPD). The results were identical with those of the previously used 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitrobluetetrazolium chloride (NBT) system. The use of AMPPD is more rapid and allows the repeated rehybridization of the membrane-bound DNA.

Base Sequence↗
PubMed1991

[Novel PCR methods for detection of HIV proviruses using digoxigenin labelled probes--Southern blot and dot blot].

Polymerase chain reaction (PCR) is useful for diagnosis of HIV infection in the silent stage, for HIV transmission from mother to child and for existence of HIV in blood and blood products. We developed a simple, rapid and safe PCR method by using digoxigenin labelled probes. Furthermore, we developed a semi-quantitative dot blot method. These methods will be helpful for diagnosis.

Blotting, Southern↗
PubMed1991

[Novel PCR methods for detection of HIV proviruses using digoxigenin labelled probes--filter paper method].

We have reported novel methods for screening of HIV by using polymerase chain reaction (PCR) and digoxigenin labelled probes. Here we developed a filter paper method for samples from long distances. About 50 microliters of peripheral bloods were absorbed in filters (5 mm diameter), dried, heated, then stored for 1 month at room temperature before PCR. This method was proved to be sufficient for detection of HIV proviruses from clinical samples.

DNA Probes↗