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PubMed1994

Flow cytometric immunodetection of human immunodeficiency virus type 1 proviral DNA by heminested PCR and digoxigenin-labeled probes.

PCR is the most sensitive and direct method for detecting blood-borne viruses, as well as an efficient means for producing vector-free probes. However, the application of PCR, especially in the laboratory diagnosis of human immunodeficiency virus (HIV) infection, is impeded by the current use of radiolabeled oligonucleotide probes. Therefore, we have developed a nonisotopic PCR immunoreactive bead (PCR-IRB) assay to detect HIV type 1 proviral DNA from peripheral blood mononuclear cells (PBMC). We used a biotinylated primer in a set of three oligonucleotides selected from the HIV long terminal repeat region for heminested PCR amplification. An internal probe was synthesized by PCR with incorporation of digoxigenin-labeled dUTP. After solution hybridization of the probe with PCR-amplified products (amplicons), the hybridized DNA was captured with streptavidin-coated magnetic beads. For the detection of hybrids, flow cytometric analyses were carried out by two procedures: (i) direct detection with fluorescein isothiocyanate (FITC)-labeled antidigoxigenin immunoglobulin G (IgG) antibody and (ii) indirect detection with antidigoxigenin sheep IgG antibody followed by FITC-labeled anti-sheep IgG antibody. Both procedures in the PCR-IRB assay detected two to three copies of HIV proviral DNA sequences, a sensitivity that is comparable with that of the conventional radioactive detection of amplicons following probe hybridization and electrophoresis. To compare the PCR-IRB assay with the conventional method, we tested 53 pedigreed PBMC specimens from blood donors and newborns; the results obtained were identical. This nonisotopic PCR-IRB assay can also be automated for potential application in laboratory diagnosis of HIV infection, blood bank screening, and therapeutic monitoring of viremia and perinatal transmission.

Base Sequence↗

PubMed1990

Rapid screening for B19 parvovirus DNA in clinical specimens with a digoxigenin-labeled DNA hybridization probe.

A rapid dot blot hybridization assay for the detection of B19 parvovirus DNA in human sera was developed. Small portions of four serum samples were mixed, filtered onto a nylon membrane, and hybridized with a digoxigenin-labeled DNA probe; for each membrane, 380 serum samples could be tested. When a dot was positive by the hybridization assay, the four serum samples dotted together were separately tested to identify the sample positive for B19 DNA. A total of 10,150 serum samples submitted for viral serological and laboratory investigation with no specific requests for B19 testing were analyzed. Nine serum samples were positive for B19 DNA by dot blot hybridization assay, and the results were confirmed by electron microscopy. This method has proven to be reliable, economical in terms of time and costs, and useful for large-scale screening of clinical specimens, both for diagnostic work and for a source of antigen.

DNA Probes↗

PubMed1993

Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes.

A polymerase chain reaction (PCR) technique for the identification of Shiga-like toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.

Antibodies, Monoclonal↗

PubMed1993

Use of digoxigenin-labelled oligonucleotide DNA probes for VT2 and VT2 human variant genes to differentiate Vero cytotoxin-producing Escherichia coli strains of serogroup O157.

Digoxigenin-labelled oligonucleotide DNA probes specific for B-subunit genes of Vero cytotoxin 2 (VT2) and a variant of VT2 (VT2vha) were used to differentiate 116 strains of Escherichia coli serogroup O157 belonging to phage types 1, 2, 4, 8, 14, and 49. Of these strains, 38% had sequences for both VT2 and VT2vha, 38% had sequences for VT2 only, and 24% had sequences for VT2vha only. Oligonucleotide probe hybridization subdivided strains of all of the phage types except phage type 1. The greatest variation in toxin gene pattern was observed with strains of phage type 14, for which there were six distinct patterns when the presence or absence of VT1 genes was also considered. Two strains from each phage type group were examined for bacteriophages encoding VT production. Two of the six VT2vha-producing strains carried phage from which DNA hybridized with the VT2vha-specific probe. Phages were not detected in the remaining four VT2vha strains, suggesting that genes may be chromosomally located or associated with a defective prophage. In contrast, seven of the eight VT2 strains carried phages from which DNA hybridized with the VT2-specific probe. Two strains of E32511 (O157:H-) were also investigated. One strain (E32511A) possessed gene sequences for both VT2 and VTvha and was shown to carry phage possessing gene sequences for VT2. With strain E32511B, however, phages were not detected and DNA hybridized only with the VT2vha probe. Analysis of total genomic DNA digested with restriction endonuclease EcoRI showed that polymorphisms were seen with VT2 strains and not with VT2vha strains.

Bacterial Toxins↗

PubMed2001

Binary typing of Staphylococcus aureus strains through reversed hybridization using digoxigenin-universal linkage system-labeled bacterial genomic DNA.

A novel binary typing (BT) procedure, based on reversed hybridization of digoxigenin-universal linkage system-labeled bacterial DNA to strip-immobilized probes, is presented. Chromogenic detection of hybrids was performed. Staphylococcus aureus isolates (n = 20) were analyzed to establish the feasibility of BT. A technically simple and fast procedure has been developed for application in routine microbiology laboratories.

Bacterial Typing Techniques↗

PubMed1990

In situ hybridisation with digoxigenin-labelled DNA probes for detection of viral genomes.

The applicability of a recently developed non-radioactive DNA labelling and detection method, which uses the digoxigenin (DIG) enzyme linked immunosorbent assay (ELISA) system, for the detection of viral infections in pathology specimens by in situ hybridisation, was examined. Its efficacy was compared with that of biotin and radioisotope labelling methods. Three cases of progressive multifocal leucoencephalopathy, two of verruca vulgaris, and seven cases of laryngeal papilloma were studied. The sensitivity of the DIG labelled probe was almost the same as that of a 35S-labelled probe in the dot-blot hybridisation test. Using in situ hybridisation with 35S-labelled and DIG labelled probes, the levels of the hybridised signals detected were similar. The biotin labelled probe was less sensitive, particularly in the cases of laryngeal papilloma. The DIG labelling and detection method was highly sensitive and applicable to the detection of viral infection by ISH, and is preferable to a radiolabelled probe, especially when in situ hybridisation is done in the pathology laboratory.

Biotin↗

PubMed1993

Demonstration of mRNA using digoxigenin labelled oligonucleotide probes for in situ hybridisation in formamide free conditions.

The value of formamide for use in in situ hybridisation (ISH) for the detection of mRNA, using either single or multi probe cocktails of digoxigenin labelled oligoprobes, was investigated. Three peptides with cell specific localisation in three separate tissues--calcitonin in the thyroid, epidermal growth factor (EGF) in the submaxillary gland, and insulin in the pancreas were studied. In each case localisation was confined to the appropriate cell type, but in the presence of formamide higher concentrations of probe and a longer development time were required. The abolition of formamide from the protocol for ISH makes the technique safer, cheaper, faster and more suitable for routine diagnostic use.

Animals↗

PubMed1997

A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation.

AIM: To determine the optimum form of labelling and the most efficient reporter molecule for non-radioisotopic in situ hybridisation (ISH). METHODS: Nine deoxyoligonucleotides complementary to histone mRNA were synthesised and labelled either enzymatically or during solid-phase synthesis with the reporter molecules digoxigenin, 2,4-dinitrophenyl (DNP), or alkaline phosphatase. Pooled deoxyoligonucleotide cocktails were then used in non-radioisotopic ISH detection of histone mRNA in human tonsil. Hybrid detection was by nitroblue tetrazoleum/5-bromo-4-chloro-3-indolyl phosphate colorimetric development. RESULTS: The use of a spacer in 3' enzymatic labelling and when labelling with alkaline phosphatase significantly increased ISH signal. The 3' and 5' labelling of oligonucleotides with triple DNP groups during solid-phase synthesis produced the strongest signal as determined by the highest cell signal intensity and shortest development time. CONCLUSIONS: 3' and 5' solid-phase labelling with triple DNP groups produced the best labelling for non-isotopic ISH using deoxyoligonucleotide cocktails.

2,4-Dinitrophenol↗

PubMed1994

Detection of bovine virus diarrhoea virus RNA by in situ hybridisation with digoxigenin-labelled riboprobes.

A non-isotopic in situ hybridisation (ISH) method has been developed for the detection of bovine virus diarrhoea virus (BVDV) RNA. The technique is highly specific and allows the detection of BVDV in formalin-fixed paraffin-wax-embedded sections. Digoxigenin-labelled riboprobes were generated to the coding region for the p125 non-structural protein and consequently it was possible to locate the virus positive-sense RNA in BVDV-infected tissues. Formalin-fixed tissues are ideal for ISH, combining the possibility of a retrospective analysis of tissues for virus distribution with excellent morphological preservation. In situ hybridisation has facilitated a precise examination, at the cellular level, of viral nucleic acid distribution during pestiviral infections, with an increased sensitivity and wider applicability than immunocytochemistry.

Animals↗

PubMed1993

DNA typing with digoxigenin-11-dideoxyuridinetriphosphate-labelled oligonucleotide probes enables non-radioactive analysis using a dual detection system: application for screening HLA-DQA1 polymorphisms.

We describe a standardized, highly sensitive, nonradioactive detection procedure for HLA class-II typing of DQA1 alleles and suballeles which has important and cost-effective application in studying HLA disease associations. The procedure involves polymerase chain reaction-based target DNA sequence amplification and dot blotting followed by stringent hybridization with digoxigenin-11-dideoxyuridine triphosphate 3'-end-labelled allele-specific oligonucleotide probes. The dual detection system described here makes this procedure very versatile.

Alleles↗

PubMed2003

Detection of oligodendrocytes in tissue sections using PCR synthesis of digoxigenin-labeled probes.

Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues.

Animals↗

PubMed1997

Demonstration of apoptotic cells in tissue sections by in situ hybridization using digoxigenin-labeled poly(A) oligonucleotide probes to detect thymidine-rich DNA sequences.

The recognition of apoptotic cells by morphological appearance alone may be difficult. We have investigated the use of in situ hybridization (ISH) with digoxigenin-labeled poly(A) probes to detect apoptotic cells in tissue sections. This method was compared to conventional morphologic assessment and in situ end-labelling (ISEL) in a range of tissues in which apoptosis is known to occur. ISH with poly(A) probes detected apoptotic nuclei in all tissues in which there was evidence of apoptosis as judged by conventional histology. ISH and, to a lesser extent, ISEL preferentially labeled shrunken but still intact nuclei with margination of chromatin, presumably at an early stage of apoptosis. The poly(A) hybridization was abolished by pretreatment of tissue sections with DNAse. After denaturation of DNA, poly(A) hybridized to nuclei in all cells. No convincing hybridization signal was detected in alcohol-fixed or fresh-frozen sections. Both ISEL and ISH labeled some of the nuclei in ischemic tissues. ISH with poly(A) oligonucleotide probes offers a simple alternative to ISEL for detection of cells in early stages of apoptosis. These probes hybridize to thymidine-rich sequences of DNA in the highly repeated Alu sequences within the nuclear genome. These sequences are believed to become available for hybridization after formalin fixation and paraffin embedding as a result of the apoptosis-related increase in the susceptibility of nuclear DNA to denaturation.

Apoptosis↗

PubMed1997

Biotin and digoxigenin as labels for light and electron microscopy in situ hybridization probes: where do we stand?

Biotin was recently applied to detect cellular DNA or RNA. In combination with avidin, streptavidin or antibody, it can be conjugated with fluorescent dye, enzyme, ferritin, or gold. However, emphasis has recently been placed on the false-positive results that are obtained when this probe is used, because endogenous biotin may sometimes interfere with specific signals. Digoxigenin appears to be an interesting alternative because it is present exclusively in Digitalis plants as a secondary metabolite. We discuss in this review the efficiency and the respective advantages and disavantages of these two probes for in situ hybridization, mainly at the electron microscopic level.

Biotin↗

PubMed1997

Detection of phosphatidylinositol glycan class A gene transcripts by RT in situ PCR hybridization. A comparative study using fluorescein, Texas Red, and digoxigenin-11 dUTP for color detection.

Gene-specific probes labeled with fluorescein, Texas Red, and digoxigenin-11 dUTP (DIG) were used for RT in situ PCR hybridization to detect PIG-A gene (phosphatidylinositol glycan class A) transcripts. The PIG-A gene is responsible for biosynthesis of the glycosylphosphatidyl-inositol (GPI) anchor. Lack of GPI anchor expression due to mutations can cause an acquired clonal hematologic disorder called paroxysmal nocturnal hemoglobinuria (PNH). In this RT in situ PCR study, two types of labeling methods, a direct method (using fluorescein and Texas Red) and an indirect method (using DIG-11 dUTP) were compared. Both were successfully applied to detect and localize the PIG-A gene transcripts within single cells of the cell lines AA2, H9, and JY. Furthermore, similar results for sensitivity and reproducibility were obtained. Advantages and disadvantages of the different labeling techniques are discussed. In addition, peripheral blood mononuclear cells from PNH patients were also included in this study.

Cells, Cultured↗

PubMed1998

Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ hybridization using CARD amplification.

A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.0-8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of the TNP-tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single- and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a variety of tyramide conjugates in bulk amounts at relatively low cost.

Biotin↗

PubMed2002

Tissue distribution of avian infectious bronchitis virus following in ovo inoculation of chicken embryos examined by in situ hybridization with antisense digoxigenin-labeled universal riboprobe.

Chicken embryos were inoculated with 8 different strains of infectious bronchitis virus (IBV) representing 7 different serotypes at 17 days of embryonation. At 2 and 5 days postinfection (dpi), tissues were collected for in situ hybridization using an antisense digoxigenin-labeled riboprobe corresponding to the sequence of the mRNA coding for the membrane protein. Extensive antigen staining in the cytoplasm of epithelial cells in the trachea, lung, bursa, and intestine was detected at 2 dpi with all 8 strains of IBV. At 5 dpi, little or no positive staining was observed in these tissues. However, tubular cells of the kidney showed multifocal positive staining with the Wolgemuth strain-, Gray strain-, JMK strain-, and Mass41 strain-infected chickens. No viral RNA was detected in the spleen at any time point. The results demonstrated strict epitheliotropic nature and wide tissue tropism of strains of IBV in the chicken embryo and the universality of our riboprobe. In situ hybridization with this probe will be useful for understanding the tissue tropism and the pathogenesis of IBV in vivo.

Animals↗

PubMed1993

Use of a digoxigenin-labeled RNA probe to detect all 24 serotypes of bluetongue virus in cell culture.

A digoxigenin-labeled RNA probe, corresponding to the section of the bluetongue virus (BTV) serotype 17 genome coding for nonstructural protein-1 (NS1), was applied to noninfected cell cultures and cell cultures infected with 24 different serotypes of BTV, 2 serotypes of epizootic hemorrhagic disease virus, and African horse sickness virus type 4. The probe hybridized to all cell cultures infected with the various BTV serotypes but did not hybridize to noninfected cell cultures or cell cultures infected with any of the other orbiviruses.

Animals↗

PubMed1994

Identification of African horse sickness virus in cell culture using a digoxigenin-labeled RNA probe.

A digoxigenin-labeled RNA probe was synthesized from a plasmid containing a portion of the African horse sickness virus (AHSV) serotype 4 genome segment coding for nonstructural protein 1. In an in situ hybridization procedure, this probe hybridized successfully to Vero cells infected with each of the 9 serotypes of AHSV. There was no hybridization with noninfected cell cultures or cell cultures infected with bluetongue virus.

African Horse Sickness Virus↗