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PubMed1986

Digoxin and metabolites in urine and feces: a fluorescence derivatization--high-performance liquid chromatographic technique.

A high-performance liquid chromatography method is described for the determination of digoxin and its metabolites digoxigenin, digoxigenin monodigitoxoside, digoxigenin bis-digitoxoside and dihydrodigoxin (20S and 20R) excreted in urine and feces. The urine sample or fecal supernatant is extracted with methylene chloride in the presence of digitoxigenin or digitoxin as internal standard. Pre-column derivatization is achieved using 1-naphthoyl chloride with subsequent separation of the derivatized compounds on either a normal- or reversed-phase system with fluorescence detection. Recoveries for digoxin and all metabolites from fecal samples were in the range 60-74%, which is comparable to that previously determined for urine samples. Standard curve data revealed linearity over a wide range of concentrations. Coefficients of variation for the analysis were less than 10% for all compounds over a range of 5-125 ng per ml urine and 10-250 ng per 200 mg feces. Peaks for digoxin and metabolites in urine and feces were obtained when human excreta were analyzed.

Chromatography, High Pressure Liquid↗
PubMed1997

A colorimetric PCR-enzyme immunoassay to identify hantaviruses.

BACKGROUND: Hantaviruses cause two serious human diseases: hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome. At least nine hantaviruses are known to be pathogenic for humans and numerous others, with unknown disease potential, have been detected in rodents. Assays to quickly identify specific hantaviruses would be useful both for clinical diagnosis and in risk assessment studies. OBJECTIVES: The goal of our study was to develop and test a specific and sensitive PCR-based assay for identification and differentiation of hantaviruses. STUDY DESIGN: We developed an assay that combined RNA-PCR amplification and colorimetric enzymatic detection to identify representative European, Asian, and north American hantaviruses. RNAs from 18 hantavirus strains of nine species were amplified in the presence of digoxigenin-dUTP by using a single pair of oligonucleotide primers and polymerase chain reaction (PCR) performed by using rTth DNA polymerase. Digoxigenin-labeled PCR products were hybridized in solution to virus type-specific biotinilated probes, captured onto streptavidin-coated microtiter plates and detected by horseradish peroxidase-labeled anti-digoxigenin antibodies and a chromogenic substrate. RESULTS AND CONCLUSIONS: The assay correctly identified each homologous virus type tested. The detection limit of the assay was approximately 15 PFU or at least 50 copies of the viral genome. The assay is simple and strain-specific and is adaptable for automation, making it more practical than other available techniques for accurate and reliable diagnosis and typing of hantaviruses.

Orthohantavirus↗
PubMed1999

Development of recombinant B subunit of Shiga-like toxin 1 as a probe to detect carbohydrate ligands in immunochemical and flowcytometric application.

Measurement of carbohydrate binding activity of Escherichia coli Shiga-like toxin in a simple and quantitative way is an important step for evaluation of antibodies with therapeutic value and of effectiveness of vaccine treatment. We constructed a plasmid vector (pVT1-B5) to express carbohydrate binding (B) subunit of Shiga-like toxin 1 without expression of toxic (A) subunit, and established a simple method to purify the recombinant B subunit, which was then labeled with digoxigenin. The binding specificity of the digoxigenin-labeled B subunit for globotriaosylceramide was established by thin-layer chromatography immunostaining. We developed an enzyme-linked immunosorbent assay using immobilized glycolipids, demonstrating high sensitivity and clear-cut specificity of the assay. The digoxigenin-labeled B subunit was also readily applicable to the detection of cell surface carbohydrate ligands by flow cytometry.

Carbohydrates↗
PubMed1997

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk.

We have developed a quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). The designed primers permitted the amplification of a 147 bp DNA fragment from a wide selection of bacteria which may grow in milk at refrigeration temperatures. Amplified PCR products generated using a digoxigenin-labelled primer were heat-denatured before being quantified by an enzyme-linked immunosorbent assay (ELISA). A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbency differences when assaying milk samples containing bacteria in the range 10(3)-10(7) cfu ml-1. The detection threshold for the PCR-ELISA assay developed in this work is 103 cfu ml-1.

Animals↗
PubMed2000

Vasopressin and oxytocin neurones of hypothalamic supraoptic and paraventricular nuclei co-express mRNA for Type-1 and Type-2 corticotropin-releasing hormone receptors.

The presence of corticotropin-releasing hormone (CRH) receptors type-1 (CRHR-1) and type-2 (CRHR-2alpha) in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, and the effects of i.c.v. injection of CRH and urocortin on arginine vasopressin (AVP) and oxytocin release, have suggested that CRH ligands have a role in osmoregulation. In this study, double labelling in situ hybridization using 35S-labelled CRHR-1 or CRHR-2alpha and digoxigenin-labelled AVP, oxytocin or CRH riboprobes was employed to examine the localization of CRHR-1 or CRHR-2alpha mRNA in the SON and PVN of control and osmotically stimulated rats. Rats received an i.p. hypertonic saline (1.5 M) injection or isotonic saline injection (controls), or 2% NaCl intake (salt loading) or tap water (controls) for 12 days. While CRHR-1 mRNA was undetectable in the SON and PVN in control rats, its expression was increased markedly at 4 h after i.p. hypertonic saline injection or after 12 days salt loading. Of the cells labelled with digoxigenin-AVP, 53% in the SON and 90% in the PVN coexpressed CRHR-1 mRNA after i.p. hypertonic saline injection. In oxytocinergic neurones, 73% in the SON and 91% in the PVN showed CRHR-1 autoradiographic grains higher than background levels after i.p. hypertonic saline injection. In addition, i.p. hypertonic saline induced CRHR-1 mRNA expression in digoxigenin-CRH stained cells in the parvocellular PVN. CRHR-2alpha transcripts were present in both the SON and PVN under basal conditions, and salt loading, but not acute i.p. hypertonic saline injection, further stimulated this expression. Double labelling in situ hybridization showed colocalization of CRHR-2alpha mRNA with AVP and oxytocin mRNA in the SON. These studies support a role for CRH and urocortin regulating the hypothalamo-neurohypophyseal system, and suggest a direct action of the peptides in the magnocellular neurones.

Animals↗
PubMed1991

Pharmacokinetics of digoxin and main metabolites/derivatives in healthy humans.

Three healthy, young male volunteers received doses of 0.6 and 1.2 mg of specifically labelled [3H]digoxin each by intravenous (i.v.) bolus injection and oral (p.o.) administration in accordance with a randomized four-way crossover design. Plasma, urine, and feces samples were taken over an interval of 144 h after drug administration. Total radioactivity and individual radioactivity assignable to digoxin and its metabolites were measured. After i.v. administration, the mean +/- SD recovery of total radioactivity, as percent of dose, was complete, urine 81.3 +/- 2.0% and feces 17.1 +/- 2.8%. The mean recovery of digoxin and that of its metabolites in urine was digoxin 75.6 +/- 3.0%, dihydrodigoxin 2.8 +/- 1.6%, digoxigenin bisdigitoxoside 1.6 +/- 0.1%, and additional metabolites 1.5 +/- 0.3%. Judging from the metabolite data in urine and considering the 5% impurity of the administered dose, metabolism of digoxin appeared to be insignificant after i.v. administration. The total and renal clearances of digoxin were, on average, 193 +/- 25 ml min-1 and 152 +/- 24 ml min-1. The mean steady state volume of distribution was 489 +/- 73 L and the mean residence time 41 +/- 5 h. For the metabolites dihydrodigoxin and digoxigenin bisdigitoxoside the mean residence times were on average 35 +/- 9 h and 53 +/- 11 h; the renal clearances were 79 +/- 13 ml min-1 and 100 +/- 26 ml min-1. After p.o. administration, the mean recovery of total radioactivity, as percent of the dose, was also complete, urine 65.7 +/- 1.98% and feces 31.6 +/- 7.6%. The mean recovery of digoxin and that of its metabolites, as percent of dose, in urine was digoxin 51.5 +/- 11.4%, dihydrodigoxin 4.5 +/- 3.9%, digoxigenin bisdigitoxoside 1.9 +/- 0.1%, polar metabolites 5.5 +/- 3.8%, and additional metabolites 1.3 +/- 0.6%. After p.o., as compared to i.v. administration, larger amounts of all the metabolites were formed in accordance with first pass metabolism/degradation. Maximum mean plasma concentrations of 4.3 +/- 2.5 ng ml-1 and 9.5 +/- 1.1 ng ml-1 for digoxin were observed at 40 +/- 10 min after p.o. administration of 0.6 and 1.2 mg of the drug. The mean absolute bioavailability of digoxin from an aqueous solution was 0.67 +/- 0.14. Renal clearance and mean oral residence time for digoxin were on average 176 +/- 28 ml min-1 and 37 +/- 4 h after p.o. administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration, Oral↗
PubMed1998

Amplification refractory mutation system linear extension: a novel, gel-free, enzyme-linked immunoassay method for DNA genotyping.

A single synthesis cycle of the amplification refractory mutation system (ARMS) was applied to the analysis of K-ras alleles amplified by polymerase chain reaction and immobilized in streptavidin-coated microtiter plates. The ARMS cycle provided the specificity and molecular switch characteristics of a conventional ARMS assay. This allowed linear extension from an allele-specific primer and the incorporation of digoxigenin-labeled deoxyuridine monophosphate from digoxigenin-11-deoxyuridine triphosphate in the presence of the appropriate K-ras allele. Any digoxigenin-labeled deoxyuridine monophosphate substitution was then demonstrated by enzyme-linked immunoassay with colorimetric endpoint. This method is capable of detecting underrepresented acquired mutations, and this has been shown by the unambiguous detection of specific K-ras mutations in cell line DNA/normal human genomic DNA admixtures. The characterization of K-ras mutations in frozen colorectal tumor samples and histologic material is also described.

Alleles↗
PubMed1989

"Reverse" DNA hybridization method for the rapid identification of subgingival microorganisms.

A "reverse" hybridization method is described, in which whole chromosomal DNA was extracted from 10-20 colonies of "unknown" strains in pure culture and labelled with digoxigenin by a random primer technique. DNA probes were prepared from a total of 23 strains and hybridized with targets containing 100 ng purified, denatured DNA from 38 reference strains fixed to nitrocellulose. 21/23 digoxigenin-labelled DNA probes successfully detected all members of the homologous species present on filters. Probes to Fusobacterium nucleatum strains 364 and MG detected 3/4 and 1/4 members of this species, respectively; 13/23 probes were 100% specific, but cross reactions between 10 probes and DNA targets from closely related, heterologous species occurred in 15/834 possible instances. False-positive reactions that occurred between closely related species were, however, easily distinguished and did not prevent the accurate identification of probe strains. Digoxigenin-labelled probes were capable of detecting 100 pg of homologous DNA. The reverse hybridization procedure allows identification or grouping of a large number of isolates within 3 days and provides a more economical means of characterizing subgingival isolates than predominant cultivable techniques and conventional phenotypic testing. This method could be adapted for the direct identification of microorganisms in subgingival plaque samples.

Bacteria↗
PubMed1997

Detection of identical ribotypes of Porphyromonas gingivalis in patients residing in the United States, Sudan, Romania and Norway.

Porphyromonas gingivalis has been isolated from periodontitis lesions in subjects from many geographical locations. The purpose of this investigation was to determine whether similar ribotypes of P. gingivalis could be detected among strains isolated in different countries. A total of 198 isolates of P. gingivalis were obtained from 52 periodontitis patients in Boston (130 isolates), Bergen, Norway (17 isolates), Khartoum, Sudan (26 isolates), and Bucharest, Romania (25 isolates). DNA was isolated from each strain, cut separately by the restriction endonucleases KpnI and PstI. The resulting preparations were subjected to electrophoresis in a 0.8% agarose gel using a Tris-acetate EDTA buffer. Uncut lambda and a 1000-bp fragment of 16S rRNA were included as internal standards in each lane. In addition, a HindIII digest of lambda was present in a separate lane in each run. The DNA fragments were transferred to a nylon membrane by downward capillary transfer. 16S rRNA bands were detected using a 1000-kb digoxigenin-labelled probe generated by a polymerase chain reaction. At the same time, a digoxigenin-labelled probe to lambda was employed to detect the internal and molecular weight standards. The bands were detected using antibody to digoxigenin conjugated to alkaline phosphatase and chemiluminescence. The positions of the bands relative to the internal standards were determined and normalized to correct for run-to-run variations, and the molecular weight of each band was determined by comparison with standards within each gel. The resulting data for the 2 enzymes were combined and subjected to cluster analysis using an average unweighted linkage sort. In some instances, isolates that appeared to be of identical ribotype using one endonuclease gave different ribotypes using the other. Strains of P. gingivalis within a subject were usually identical, except for 3 patients who harbored 2 different ribotypes/individual. All subsequent analyses employed a single ribotype strain for each subject. A total of 32 ribotypes were observed for isolates from distant countries. A total of 11.5% of the patients had isolates exhibiting the same ribotype: ribotype 7a. Identical ribotypes of P. gingivalis can be recovered from subgingival plaque samples of periodontitis patients in different countries.

Bacterial Typing Techniques↗
PubMed1977

Hydrolysis of digoxin by acid.

Predictable hydrolysis of [3H]digoxin-12alpha occurred in vitro with incubation in HCl or gastric juice. Hydrolysis varied with pH, time, temperature and agitation. Digoxin, the bis- and mono-digitoxosides of digoxigenin and digoxigenin were separated by silica gel thin-layer chromatography using chloroform-ethyl acetate-glacial acetic acid (25:25:1 v/v) and were quantitated by liquid scintillation spectrometry. Hydrolysis with incubation at 37 degrees and pH 3 for 90 min was minimal, but increased with increasing acidity until greater than 70% was hydrolysed at pH 1-2 after 30 min and greater than 96% after 90 min incubation. At pH 0-9, 87% was hydrolysed after 30 min. In vitro hydrolysis in gastric fluid was slightly less than in HCl at the same pH. A volunteer was given 150 muCi[3H]digoxin-12alpha by nasogastric tube during a pentagastrin infusion when gastric pH was 0-94. He remained on his left side and samples were aspirated at intervals and immediately neutralized. Ethanol-chloroform 50-50 (v/v) extracts of the gastric fluid aspirated after 90 min and of all the urine specimens collected for 5 days were applied to a DEAE Sephadex LH-20 column. The radioactivity appeared in a single peak as digoxigenin in the 90 min gastric aspirate and in all urine specimens. Extensive intragastric hydrolysis of digoxin may occur under conditions of maximum acid output.

Adult↗
PubMed1986

Reduction of digoxin to 20R-dihydrodigoxin by cultures of Eubacterium lentum.

The anaerobic bacterium Eubacterium lentum, a common constituent of the intestinal microflora, inactivates digoxin by reducing the unsaturated lactone ring. Reduction of the cardiac glycoside by growing cultures of E. lentum ATCC 25559 proceeded in a stereospecific manner, with the 20R-dihydrodigoxin constituting more than 99% of the product formed. This is in contrast to the 3:1 ratio of 20R and 20S epimers formed in the chemical catalytic hydrogenation. Formation of the reduced glycosides proceeded quantitatively when an overall concentration of 10 micrograms/ml was added to the cultures. E. lentum did not hydrolyze the digitoxose sugars from C-3 of the parent glycoside. However, the synthetically prepared sugar-hydrolyzed metabolites (digoxigenin, digoxigenin monodigitoxoside, and digoxigenin bisdigitoxoside) were reduced to the corresponding dihydro metabolites. Repetition of the experiments with a feces sample from a volunteer who was known to be a converter of digoxin to dihydrodigoxin gave results identical to those obtained with pure E. lentum cultures.

Chromatography, High Pressure Liquid↗
PubMed1969

Binding of digitoxin and some related cardenolides to human plasma proteins.

Tritium-labeled digitoxin, digitoxigenin, digoxin, and digoxigenin of established purity and chemcal authenticity were used to study the binding of these compounds to human plasma proteins. 97% of digitoxin in plasma was nondialyzable. Continuous flow paper electrophoresis of plasma containing digitoxin and dialysis experiments in which human serum albumin competed for the glycoside with plasma or plasma protein fractions demonstrated that digitoxin was almost exclusively bound by albumin. Equilibrium dialyses revealed that the interaction was characterized by a single binding site on the albumin molecule and an association constant of 9.62 x 10(4) liter/mole at 37 degrees C. At 1 degrees C the association constant was 4.64 x 10(4) liter/mole. The interaction therefore was endothermic; the gain in enthalpy of 3.5 kcal/mole and the free energy change of - 7.06 kcal/mole was derived from a large change in entropy of 33.8 cal/mole per degrees K. The direction of these thermodynamic changes suggested the formation of a hydrophobic bond between digitoxin and albumin. Quenching of the fluorescence of albumin by digitoxin indicated that the conformation of albumin was altered by the binding process.Digitoxigenin, its mono- and didigitoxosides, digoxin, and digoxigenin competed with digitoxn for its binding site on albumin. The affinity of the mono- and didigitoxosides for the site was equal to that of digitoxin, but that of digitoxigenin was only one-third as great. The ability of the digitoxose residues of the glycosides to enhance binding to albumin was also observed with digoxin, which was more extensively bound by the protein than digoxigenin. At concentrations of 2 mug/ml or less in plasma, only 23% of digoxin was bound. Albumin, which interacted with digoxin with an apparent association constant of 9 x 10(2) liter/mole at 37 degrees C, was entirely responsible for the binding. Lowering the temperature from 37 degrees to 1 degrees C decreased the fraction of digoxin bound to albumin by two-thirds. The marked difference in avidity of digitoxin and digoxin for serum albumin is reflected by the higher plasma concentrations, lower rate of urinary excretion, and longer half-time of digitoxin as compared to those of digoxin when these compounds are administered to man.

Blood Protein Electrophoresis↗
PubMed1999

Electron microscopic in situ DNA nick end-labeling in combination with immunoelectron microscopy.

We describe an in situ DNA nick end-labeling method that can be performed at the electron microscopic level and can also be combined with immunoelectron microscopy. As the materials, we used skin tissues from normal skin and from Bowen's disease that had been cryofixed, freeze-substituted, and embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with a reaction buffer containing digoxigenin-dUTP and terminal deoxynucleotidyl transferase. Digoxigenin nucleotides were labeled with anti-digoxigenin antibodies conjugated with colloidal gold. Specific signals were detected in the condensed chromatin of differentiated epidermal cells and hair follicles in normal skin and of dyskeratotic cells in Bowen's disease. The labeling density over chromosomal areas of apoptotic cells was significantly higher than that over chromosomal areas of mitotic cells or cytoplasmic areas. Ultrastructure was well preserved and double staining with an anti-keratin antibody was also successfully performed. This simple method has a wide range of applications to identify the nature of apoptotic cells and explore the mechanisms of apoptosis.

Adult↗
PubMed1991

Non-radioactive detection of nerve growth factor receptor (NGFR) mRNA in rat brain using in situ hybridization histochemistry.

Radioactively labeled RNA probes in conjunction with in situ hybridization histochemistry have become a useful method for studying gene expression in the central nervous system. We used digoxigenin-labeled uridine triphosphate to synthesize cRNA probes for localization of nerve growth factor receptor (NGFR) mRNA in the rat basal forebrain. Detection of cells containing digoxigenin-labeled NGFR mRNA was accomplished using a digoxigenin antibody conjugated with alkaline phosphatase. NGFR mRNA-positive cells were distributed in three major cell groups in the basal forebrain: the medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. This technique provides a rapid and sensitive method for high-resolution detection of mRNA species in the central nervous system, as well as the potential for co-localization of two different mRNA species within individual cells.

Animals↗
PubMed2000

Preparation of specific antisera to digoxin by using digoxin C-3' and C-3" hemisuccinate-bovine serum albumin conjugates.

The preparation and antigenic properties of digoxin C-3' and C-3" hemisuccinate-bovine serum albumin (BSA) conjugates are described. The antisera were prepared by immunizing rabbits with each of the digoxin-BSA conjugates, and properties of the antisera were characterized by RIA with 3H-labeled digoxin. The antidigoxin antiserum from immunization with digoxin 3'-hemisuccinate-BSA conjugate possessed high specificity for digoxin, exhibiting fairly low cross-reactions with dihydrodigoxin (2.1%), digoxigenin monodigitoxoside (0.9%), digoxigenin bisdigitoxoside (0.6%), and digoxigenin (0.1%).

Cross Reactions↗
PubMed2005

Properties of novel anti-digoxin antisera in radioimmunoassay using homologous and site heterologous tritium-labeled antigens involving a [3H]-leucine moiety.

The specificities of antisera against digoxin C-3' or C-3'' hemisuccinate-bovine serum albumin (BSA) conjugate were assessed by cross-reactivity studies with digoxin metabolites by radioimmunoassay (RIA) using the homologous and the site heterologous tritium-labeled antigens. One of the tracers used was digoxin 3'-hemisuccinyl-[3H]-leucine; the other was digoxin 3''-hemisuccinyl-[3H]-leucine, which had been prepared from digoxin 3''-hemisuccinate. When the tracer with [3H]-leucine at the C-3' position was used, antisera (I-1, I-3) elicited by digoxin 3'-hemisuccinate-BSA conjugate showed the following cross-reactivity: digoxigenin bisdigitoxoside (0.34%, 76%), digoxigenin monodigitoxoside (0.11%, 65%), digoxigenin (0.02%, 26%) and dihydrodigoxin (9.4%, 1.2%). However, when using the homologous antigen, antiserum (I-1) was highly specific against the digitoxose chain. When the site heterologous antigen, digoxin 3''-hemisuccinyl-[3H]-leucine was combined, this antiserum showed high cross-reactivity to digoxin degradation products. This digoxin RIA using antiserum (I-1) with the homologous antigen measures unmetabolized digoxin. On the other hand, the RIA system using antiserum (I-3) with the homologous antigen had cross-reactivity with the metabolites in accordance with their relative cardio-activities, so this system would be useful in therapeutic drug monitoring of digoxin.

Animals↗
PubMed1996

Expression of alpha 2- and beta-adrenoceptor subtypes in human islets of Langerhans.

Sequences from cDNA molecules encoding alpha 2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha 2C2-, alpha 2C4- and alpha 2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of alpha 2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic beta-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both alpha 2 C2 and alpha 2 C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding alpha 2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each alpha 2-adrenoceptor subtype in sections of human pancreas. All three subtypes of alpha 2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffin-embedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three alpha 2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple alpha 2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of alpha 2-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotting of RNA extracted from the clonal beta-cell line, HIT-T15. Transcripts encoding each of the three cloned alpha 2-adrenoceptor subtypes were detected in HIT-T15 cells. Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with beta 2-adrenoceptor mRNA revealed expression of this species in islet beta-cells but not in the exocrine tissue of the pancreas.

Base Sequence↗
PubMedDate not supplied

In situ hybridization, immunohistochemistry, and in situ reverse transcription-polymerase chain reaction for detection of infectious bursal disease virus.

Development of molecular techniques for the detection of infectious bursal disease virus (IBCV) is an important area of research. An in situ hybridization (ISH) test was developed with a 491-bp cDNA fragment derived from the VP2 gene of IBDV. The fragment was amplified and simultaneously labeled with incorporation of digoxigenin-11-dUTP in a nested polymerase chain reaction (PCR) assay. The resulting digoxigenin-labeled 491-bp nested PCR product was used as probe for ISH to detect and localize IBDV RNA in formalin-fixed, paraffin-embedded bursae of Fabricius from chickens both experimentally infected as well as commercially reared. Bursae from six clinically ill commercial broilers suspected to be IBDV infected were examined by ISH and immunohistochemistry. In two samples, IBDV infection was detected by both ISH and immunohistochemistry, whereas in the other two histologically normal bursae, IBDV was detected only by ISH. Two commercial chickens with atrophied bursae were negative by both ISH and immunohistochemistry. No positive IBDV stained cells were in RNase treated sections from infected birds, uninfected chickens, or reovirus-infected chickens. The ISH test developed herein resulted in important modifications, which makes it superior to other previously published procedures. We also described a direct in situ reverse transcription-polymerase chain reaction method for the amplification and detection of IBDV genome in formalin fixed, paraffin-embedded bursae of Fabricius with a single primer pair with direct incorporation of digoxigenin-11-deoxyuraciltriphosphate (dUTP) into the amplicon. Both molecular tests with their important modifications represent improved detection of IBDV.

Animals↗