In situ detection of cytomegalovirus DNA in biopsies of AIDS patients using a hybrido-immunocytochemical assay

An in situ hybrido-immunocytochemical assay, with a digoxigenin-labelled probe, was used to show the presence of cytomegalovirus DNA in both paraffin and frozen sections from tissue blocks of 5 AIDS patients. The hybridization probe was constructed by using two different DNA fragments of the repeated sequences of the CMV genome. The CMV DNA probe hybridized in situ was immunocytochemically visualized by anti-digoxigenin Fab fragments labelled with alkaline phosphatase. This hybridization procedure proved to be sensitive, specific, and provided good resolving power. Thus, it might effectively be employed in immunohistological and virological laboratories for the diagnosis of CMV infections in AIDS patients; indeed it might even be applied further in the virological context.     

In situ detection of cytomegalovirus DNA in biopsies of AIDS patients using a hybrido-immunocytochemical assay

Summary An in situ hybrido-immunocytochemical assay, with a digoxigenin-labelled probe, was used to show the presence of cytomegalovirus DNA in both paraffin and frozen sections from tissue blocks of 5 AIDS patients. The hybridization probe was constructed by using two different DNA fragments of the repeated sequences of the CMV genome. The CMV DNA probe hybridized in situ was immunocytochemically visualized by anti-digoxigenin Fab fragments labelled with alkaline phosphatase. This hybridization procedure proved to be sensitive, specific, and provided good resolving power. Thus, it might effectively be employed in immunohistological and virological laboratories for the diagnosis of CMV infections in AIDS patients; indeed it might even be applied further in the virological context.     

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.     

Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.     

A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay

We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.     

Preparation of enzyme-conjugated DNA probe and application to the universal probe system.

A general procedure for the cross-linking of enzyme to DNA has been developed for use as a nonradioactive probe. In this method, DNA is transaminated with diaminopropane to introduce primary amino groups into the cytosine residues. Then the amino groups are converted to thiol groups using a heterobifunctional cross-linker. The thiolated DNA is conjugated with the maleimide-introduced enzyme. With this method, alkaline phosphatase was cross-linked to a single-stranded DNA (sspUCRf1). The conjugate was able to detect 5 pg of target DNA (pUCf1 plasmid, 3.2 kbp) fixed onto the nitrocellulose membrane, using a colorimetric assay. The enzyme-conjugated DNA was applied to "the universal probe system," which consisted of two single-stranded DNA probes (a primary probe and a labeled secondary probe). Using alkaline phosphatase-conjugated sspUCRf1 DNA as the secondary probe, the c-myc gene and HBV DNA were detected effectively on Southern and dot-blot hybridization.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Summary Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV 16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF-or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidinbiotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV 16 DNA and haptenized HSV2 DNA.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF- or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidin-biotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV16 DNA and haptenized HSV2 DNA.     

Evaluation of a Viral Microarray Based on Simultaneous Extraction and Amplification of Viral Nucleotide Acid for Detecting Human Herpesviruses and Enteroviruses

In this study, a viral microarray based assay was developed to detect the human herpesviruses and enteroviruses associated with central nervous system infections, including herpes simplex virus type 1, type 2 (HSV1 and HSV2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), enterovirus 71 (EV71), coxsackievirus A 16 (CA16) and B 5(CB5). The DNA polymerase gene of human herpesviruses and 5’-untranslated region of enteroviruses were selected as the targets to design primers and probes. Human herpesviruses DNA and enteroviruses RNA were extracted simultaneously by using a guanidinium thiocyanate acid buffer, and were subsequently amplified through a biotinylated asymmetry multiplex RT-PCR with the specific primer of enteroviruses. In total, 90 blood samples and 49 cerebrospinal fluids samples with suspected systemic or neurological virus infections were investigated. Out of 139 samples, 66 were identified as positive. The specificities of this multiplex RT-PCR microarray assay were over 96% but the sensitivities were various from 100% for HSV1, HSV2, EV71 and CB5, 95.83% for CMV, 80% for EBV to 71.43% for CA16 in comparison with reference standards of TaqMan qPCR/qRT-PCR. The high Kappa values (>0.90) from HSV1, HSV2, CMV, EV71 and CB5 were obtained, indicating almost perfect agreement in term of the 5 viruses detection. But lower Kappa values for EBV (0.63) and CA16 (0.74) displayed a moderate to substantial agreement. This study provides an innovation of simultaneous extraction, amplification, hybridization and detection of DNA viruses and RNA viruses with simplicity and specificity, and demonstrates a potential clinical utility for a variety of viruses’ detection.     

Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate

R. J. CANO, M.J. TORRES, R.E. KLEM, J.C. PALOMARES AND J. CASADESUS. 1992. This study evaluates a DNA hybridization assay for salmonella with A tto P hos ™ (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65°C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/1 A tto P hos ™. The reaction was evaluated after 30 min at 37°C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10 000 copies of target DNA or 5 times 10^-20 mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and A tto P hos ™, is a specific and highly sensitive quantitative method for the detection of salmonellas.     

Characterization of alkaline phosphatase labeled UidA(Gus) probe and its application in testing of transgenic <Emphasis Type="Italic">tritordeum</Emphasis>

Hybridization is a very important molecular biology technique to measure the degree of genetic similarity between DNA sequences, and detect the foreign genes in transgenic organisms. To label a DNA or RNA probe plays a key role in hybridization. A method using nonradioactive material alkaline phosphatase to label UidA(Gus) DNA as probe has been studied. On that basis of Renz and our previous work, alkaline phosphatase-labeled DNA was used as a probe to examine the transformation of the foreign UidA(Gus) gene in transgenic tritordeum. Such DNA–enzyme complexes were characterized and examined carefully, the results showed that it was a sensitive, specific, safe and economical probe. For dot hybridization and Southern blot under full-stringency conditions with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-Nitro Blue Tetrazolium (NBT) as the substrate, dot hybridization showed that the UidA(Gus) gene was transformed into the target plants and inherited stable, Southern blot showed that at least two copies of UidA(Gus) gene were inserted into one line of our transgenic tritordeum. Histochemical staining with X-Gluc of transgenic tritordeum also certified that the foreign UidA(Gus) DNA were transformed into the transgenic tritordeum.     

Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences

Conditions for combination of DNA in situ hybridization, using biotinylated DNA probes, with immunohistochemistry were investigated on cryostat sections, cytological preparations, and paraffin sections. We found that cryostat sections and cytological preparations are suitable for in situ hybridization of target DNA after fixation in acetone, methanol, ethanol, or Carnoy without further proteinase pretreatment. Acetone is also very suitable for immunostaining of cell surface or cytoskeleton antigens. We therefore performed combined immunoenzyme and in situ hybridization staining using this fixative. The best results were obtained when immunoperoxidase staining with diaminobenzidine/H2O2 was followed directly by in situ hybridization. In addition to immunoperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) staining with naphthol ASBI phosphate and New Fuchsin as a substrate could be used. In most instances, detection of the biotinylated hybrid with a streptavidin-biotinylated polyalkaline phosphatase method using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as the substrate was preferable. The double stainings were studied on the following test models: (a) frozen tonsil sections: cell surface antigens (pan T) and ribosomal DNA; (b) frozen genital condyloma sections; cytokeratins and human papillomavirus type 6 + 11 (HPV-6/11) DNA; (c) CaSKi cells: cytokeratins and HPV-16 DNA; (d) infected fetal lung fibroblasts: vimentin and cytomegalovirus (CMV) DNA. An adapted procedure was followed on routinely formaldehye-fixed and paraffin-embedded condyloma tissue. Immunoperoxidase staining for papilloma virus capsid antigen could be combined with DNA in situ hybridization with HPV-6/11 DNA. In this model, however, the accessibility of the target DNA had to be improved by enzyme treatment after the immunostaining and before starting the in situ hybridization.     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

Non-isotopical labeling of murine heterochromatin in situ by hybridization with in vitro-synthesized biotinylated gamma (major) satellite DNA

Degenerate probe DNA, homologous to part of the 234-bp repeated mouse gamma (major) satellite DNA, was generated by primer-directed in vitro DNA amplification using the polymerase chain reaction with oligonucleotide primers that anneal in the most conserved parts of the repeat. Probe labeling with biotin was performed during DNA polymerization. In situ hybridization of probe DNA with metaphase chromosome preparations showed exclusive binding of probe molecules to the centromeric region of mouse chromosomes. We applied the probe DNA for labeling of mouse heterochromatin in metaphase chromosomes, as well as interphase cell nuclei, and compared results of probe visualization using avidin tagged with either fluorescein or alkaline phosphatase in combination with a chromogenic substrate.     

A comparison of substrates for quantifying the signal from a nonradiolabeled DNA probe.

A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described. In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase. Four substrates [4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)] were assessed for use in a quantitative hybridization assay. Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg. In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P. perfectomarina genomic DNA and total RNA. The amount of hybridized probe was determined using AttoPhos. Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA. Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg. This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.     

Use of oligonucleotide-alkaline phosphatase conjugates as non-radioactive probes for rapid analysis of a proteinase inhibitor gene fromZea mays

Conjugates of oligonucleotides and alkaline phosphatase have been prepared and used as nonradioactive hybridization probes for the study ofPis3 (=MPI) a gene encoding a proteinase inhibitor fromZea mays. Attachment of the alkaline phosphatase was carried out either at the 5′ or 3′ end of two 25-bp oligonucleotides. Sensitivity of each alkaline phosphatase-oligonucleotide probe was assessed using a chemiluminescent substrate for detection of alkaline phosphatase activity. This sensitive method allows the rapid analysis of genomic clones isolated from aZea mays library and the subsequent characterization of the completePis3 gene without the need for construction of restriction maps for the cloned DNA fragments. This general strategy may be valuable for the identification of any gene for which a limited sequence is known and for location of specific DNA sequences that represent a small region within a larger DNA fragment.     

The isolation and partial sequencing of human bone alkaline phosphatase gene

1. A charon 4A human fetal liver genomic library was screened for human alkaline phosphatase sequences using the cloned human bone cDNA as a hybridization probe. 2. A positive clone was obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing.     

Schistosoma mansoni: chromosomal localization of DNA repeat elements by in situ hybridization using biotinylated DNA probes

Localization of the SM alpha family of repeated DNA and the rDNA repeat on the chromosomes of Schistosoma mansoni by in situ hybridization is presented. Biotinylated DNA was hybridized to target chromosomes and hybridization was detected using either alkaline phosphatase-labeled avidin or fluorescein-labeled avidin and biotinylated anti-avidin antibody. Hybridization detection using a fluorescein conjugate was more specific and sensitive with less background noise than detection with alkaline phosphatase conjugates. SM alpha hybridizing sequences were found dispersed throughout the genome, hybridizing to the sex chromosomes and autosomes. The SM alpha probe showed specific hybridization to the euchromatic gap region within the large heterochromatic block of the short arm of the W chromosome. This specific hybridization coupled with the lack of chiasma formation in this region of the ZW bivalent (presumably due to the heterochromatinization of this region) may explain the pattern of sex-specific hybridization reported for the SM alpha family. The rDNA repeat was localized to the secondary constriction of the short arm of chromosome 3. Specifically, the rDNA probe hybridized with the stalk of the secondary constriction and with parts of both side regions, the satellite and the short arm proper.     

Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.

Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated.