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.     

Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.     

Influence Factors of in Situ Hybridization in Triticeae

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post-hybridization washing in in situ hybridization were optimized. The results are as fel-lows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method: whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75 ℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2 × SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post-hybridization washes were carried out in 50% formamide (in 2 × SSC) or in 2 × SSC at different temperature. When the post-hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2 × SSC). specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.     

Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes

 Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level. Accepted: 16 February 1996     

Use of non-radioactive DNA probes for the characterization of adult T-cell leukemia cells

DNA fragments were labeled with dinitrophenyl (DNP) residues by the reaction with 2,4-dinitrobenzaldehyde in alkaline condition and the labeled DNA was used as a probe for non-radioactive in situ hybridization. DNP-labeled DNA probes for T cell receptor beta chain, c-myc and HTLV-1 were hybridized in situ to mRNA on cell specimens fixed with Carnoy's fixative. DNA-mRNA hybrids were detected immunohistochemically using anti-DNP antibodies. Cytoplasms of adult T cell leukemia cells were stained with varied intensity when these probes were used. More than 70% of cells were positively stained with T cell receptor probe. However, less than 30% of cells were stained with c-myc and HTLV-1 probes. The present study indicates that non-radioactive in situ hybridization can be used for the characterization and classification of leukemia.     

An improved method for chromosome-specific labeling of alpha satellite DNA in situ by using denatured double-stranded DNA probes as primers in a primed in situ labeling (PRINS) procedure.

An improved primed in situ labeling (PRINS) procedure that provides fast, highly sensitive, and nonradioactive cytogenetic localization of chromosome-specific tandem repeat sequences is presented. The PRINS technique is based on the sequence-specific annealing in situ of unlabeled DNA. This DNA then serves as primer for chain elongation in situ catalyzed by a DNA polymerase. If biotin-labeled nucleotides are used as substrate for the chain elongation, the hybridization site becomes labeled with biotin. The biotin is subsequently made visible through the binding of FITC-labeled avidin. Tandem repeat sequences may be detected in a few hours with synthetic oligonucleotides as primers, but specific labeling of single chromosomes is not easily obtained. This may be achieved, however, if denatured double-stranded DNA fragments from polymerase-chain-reaction products or cloned probes are used as primers. In the latter case, single chromosome pairs are stained with a speed and ease (1 h reaction and no probe labeling) that are superior to traditional in situ hybridization. Subsequent high-quality Q banding of the chromosomes is also possible. The developments described here extends the range of applications of the PRINS technique, so that it now can operate with any type of probe that is available for traditional in situ hybridization.     

Non-radioactive in situ hybridization for the detection of cytomegalovirus infections

Summary Acetylaminofluorene (AAF) modified cytomegalovirus (CMV) DNA probes have been applied for the rapid detection of CMV genomes by non-radioactive in situ hybridization in routinely obtained pathological material. To establish proper protocols, AAF modified mouse satellite DNA and mouse liver were used to investigate the procedural variables. Among these were type and time of fixation, glass slide coating for improved tissue adherence, protease permeabilization of sections, type and time of denaturation and hybridization, probe concentration, post-hybridization washing conditions and immunocytochemical detection. This research has led to a user-friendly procedure which, in addition to cells displaying a cytopathological effect typical for CMV infection detects with high sensitivity CMV carrying cells that show no histo-pathological alterations. It can be readily applied in routine clinical-diagnostic laboratories.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySubsidized in part by Het Praeventiefonds, The Hague, The Netherlands (project no. 28-801)     

A polymorphic DNA marker genetically linked to congenital malformations

Unusual restriction fragments were detected by DNA blot hybridization with PCNA (DNA polymerase-delta auxiliary protein) probe in one of seven cases of congenital malformations. Chromosomal in situ hybridization localized PCNA gene to region q31-35 of human chromosome 2. To discover the locus more closely associated with congenital malformations, a cloned DNA segment which has been mapped to chromosomal region 2q33-36 was tested for restriction fragment length polymorphisms (RFLPs) in these patients. The 2q33-36 probe hybridized with 2.1-kb, 1.9-kb and 1.7-kb fragments in ten normal control samples. In seven cases of congenital malformations examined, however, the band of 2.1 kb is absent in six cases and the band of 1.7 kb in one case. These results indicate that the locus closely linked to congenital malformations is present in the proximity of PCNA locus.     

Non-radioactive in situ hybridization for the detection of cytomegalovirus infections

Acetylaminofluorene (AAF) modified cytomegalovirus (CMV) DNA probes have been applied for the rapid detection of CMV genomes by non-radioactive in situ hybridization in routinely obtained pathological material. To establish proper protocols, AAF modified mouse satellite DNA and mouse liver were used to investigate the procedural variables. Among these were type and time of fixation, glass slide coating for improved tissue adherence, protease permeabilization of sections, type and time of denaturation and hybridization, probe concentration, post-hybridization washing conditions and immunocytochemical detection. This research has led to a user-friendly procedure which, in addition to cells displaying a cytopathological effect typical for CMV infection, detects with high sensitivity CMV carrying cells that show no histo-pathological alterations. It can be readily applied in routine clinical-diagnostic laboratories.     

A single-step silver enhancement method permitting rapid diagnosis of cytomegalovirus infection in formalin-fixed, paraffin-embedded tissue sections by in situ hybridization and immunoperoxidase detection

A single-step silver enhancement method was developed which intensifies the polymerized nickel-complexed diaminobenzidine (Ni-DAB) reaction product of peroxidase. With such enhancement, an in situ hybridization procedure can be performed in less than 8 hr by using a 2-hr hybridization incubation and direct detection. Cytomegalovirus (CMV)-infected lung sections were hybridized in situ for 2 hr with a biotinylated CMV genomic-length probe. The probe was detected directly with avidin-biotinylated peroxidase using Ni-DAB as the substrate, and the reaction product was enhanced with silver. Silver was deposited only on the Ni-DAB and not on normally argyrophilic substances. Indirect detection of the probe using a sandwich technique before silver enhancement proved more sensitive, but the length of the procedure was increased without substantially changing the result (infection vs. no infection). Sensitivity was also improved by omitting the dehydration step before applying the probe, and by increasing the temperature and duration of denaturation. In a blinded study of 21 open-lung biopsies, 13 of 13 culture-negative specimens were negative by hybridization, and 7 of 8 culture-positive specimens were positive by hybridization. Modified short hybridization with a biotinylated probe and silver-enhanced direct detection therefore provides a rapid but sensitive method for diagnosis of viral infection.     

Direct diagnostic methods for the demonstration of cytomegaloviruses in the blood

CMV can be detected in blood with two techniques: isolation of CMV from the buffy-coat on human embryonic fibroblasts; CMV genome detection by DNA hybridization technique. Isolation of CMV from blood plated on embryonic fibroblasts necessitates a delay of 10-30 days. Using monoclonal antibodies we have developed a 96 hours test to diagnose viremia. Fibroblasts grown on coverslip in tubes are inoculated with buffy-coat. Indirect immunofluorescence test is performed using the monoclonal antibodies E 13, 48 and 96 hours post-infection. DNA-DNA hybridization technique necessitates purification of CMV DNA. CMV DNA is then labelled with 32 P by nick-translation. This labelled DNA is used as probe for detection of CMV genome in DNA extracted from leukocytes. The hybridization is virus-specific. Cytomegaloviremia correlates with active infection and is not indicate of the carrier state of blood donors. CMV genome research in blood can potentially identify infective donors. Further studies are needed to correlate CMV genome detection in blood with presence of CMV antibodies.     

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.     

间接核酸杂交方法的建立

建立了一种新的核酸杂交手段一间接核酸杂交方法,其突出的优点是用一种共同的核酸标记物就可检查不同的基因组或不同的基因。我们重组乙型肝炎病毒或EB病毒的核酸片段于噬菌体M_(13)mp8载体,以此重组的单链DNA为第一夹心层,用~(32)P标记的双链噬菌体DNA作为共同探针,检查乙型肝炎病毒和EB病毒的核酸,获得满意的结果。应用该法进行细胞内的原位杂交,检查细胞内存在的EB病毒基因效果亦佳。     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome l-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using β-galactosidase/ ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. the described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.     

The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.     

Pig genome analysis: differential distribution of SINE and LINE sequences is less pronounced than in the human and mouse genomes

The distribution of SINE and LINE sequences in the pig genome was examined by fluorescence in situ hybridization (FISH), interspersed repeat PCR, and restriction analysis of high molecular weight DNA. FISH revealed a largely uniform hybridization to the euchromatic chromosome regions with both interspersed repeats, although a bias toward the G-bands was observed for the LINE probe. Southern blots of inter-SINE and inter-LINE PCR products showed strong hybridization to LINE and SINE probes, respectively. High molecular weight DNA derived from a pig |m~ hamster hybrid cell line was cut with a panel of G + C and A + T rich rare cutter restriction enzymes, then run on a pulsed field gel and Southern blotted. Sequential hybridization with SINE and LINE probes showed that SINE hybridization was to relatively low molecular weight fragments with the G + C rich enzymes, whereas the LINE probe gave hybridization to significantly larger fragments produced by these enzymes. DNA samples digested with A + T rich enzymes gave essentially similar patterns with SINE and LINE probes. We conclude that the pattern of differential distribution of SINEs and LINEs, which has been described in man and mouse, does exist in the pig but is much less pronounced. Received: 25 April 1995 / Accepted: 1 September 1995     

A DNA probe for specific detection of Mycoplasma pulmonis

Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.     

Regional localization of chromosome 3-specific DNA fragments by using a hybrid cell deletion mapping panel.

A series of human chromosome 3-specific DNA fragments isolated and characterized from a lamda phage genomic library were regionally localized on human chromosome 3. This was accomplished using filter hybridization blot analysis of a human chromosome 3 hybrid cell deletion mapping panel. Twenty-three new anonymous DNA fragments were assigned to one of four physical regions of chromosome 3. Seventeen DNA fragments were mapped to the long arm of chromosome 3, including one DNA fragment that demonstrated a restriction fragment length polymorphism (RFLP). Five DNA fragments were assigned to 3p14.2----pter, including one highly polymorphic fragment sublocalized at 3p25----pter by in situ hybridization. This DNA fragment is the second reported distal 3p polymorphic probe. One DNA fragment was localized to 3p14----p14.2. In addition, three fragments previously assigned to chromosome 3 were confirmed. Polymorphic DNA probes DNF15S2 (formerly D1S1) and D3S2 were mapped to 3p14.2----pter. The previous 3p25 in situ localization of the c-raf-1 oncogene was supported by deletion panel mapping. The physical localization of these twenty-three new DNA fragments has more than doubled the number of cloned DNA fragments assigned to chromosome 3. These and future regional assignments of DNA fragment probes will facilitate construction of both a physical and genetic linkage map of chromosome 3. They may also be useful in characterizing the chromosomal and molecular aberrations involved in small-cell lung cancer (SCLC), renal cell carcinoma, other malignancies, and the 3p14.2 common fragile site.     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.