Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue–green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140 nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0 kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e−124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue-green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e-124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

Chromosome localization of the human renin gene (REN) by in situ hybridization

Previous studies by Southern blot analysis of human X mouse somatic cell hybrids localized the renin gene to region p21----qter of human chromosome 1. Using a DNA insert encoding exons 2-5, the renin gene was mapped to human chromosome bands 1q25----q32 by in situ hybridization. The sublocalization of the renin gene will facilitate subsequent detailed linkage analysis of human chromosome 1.     

Chromosomal localization of the mouse prealbumin gene (Ttr) by in situ hybridization.

Prealbumin is a serum protein which plays an important role in plasma transport of retinol and thyroxine. The accumulation of a variant prealbumin is associated with a hereditary disorder, familial amyloidotic polyneuropathy (FAP). In situ hybridization with a mouse prealbumin gene cDNA probe was carried out in mouse fibroblasts. Analysis of 114 R-banded metaphases showed that 13% of the total grains were located on chromosome 4 and 46% of the grains on this chromosome were in the region C6-D1. Linkage and syntenic group analysis showed that the prealbumin gene (Ttr) is located between two syntenic groups on mouse chromosome 4, which corresponded to two syntenic groups present on human chromosomes 1 and 9.     

Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides

Members of the genus Dehalococcoides are well-known for their capacity to reductively dechlorinate chlorinated organic pollutants. The availability of quantitative and sensitive detection methods is of major interest for research on the ecology of those environmentally important micro-organisms. In this paper we describe the development of a Catalyzed Reporter Deposition-Fluorescent In Situ Hybridization (CARD-FISH) for detection of Dehalococcoides cells in enrichment cultures using two oligonucleotide sequences which target two different lineages of Dehalococcoides as probes. Both sequences were previously applied in conventional FISH as probes. Conjugation of the probe to horseradish peroxidase (HRP) did not change the specificity of the probes and bright fluorescent signals were obtained. Despite the use of higher concentrations of probe and the application of longer exposure times in the conventional FISH procedure, CARD-FISH resulted in more intense signals. The CARD-FISH method was applied to a vinyl chloride (VC)-reductively-dechlorinating enrichment culture. Only the probe targeting the CBDB1 lineage of Dehalococcoides reacted with the sample which was in agreement with previous nucleic acid based analysis. The culture consisted of 51%+/-8% of Dehalococcoides cells. Furthermore, the CARD-FISH probes for detecting Dehalococcoides were combined with FISH probes for simultaneous detection of either Bacteria or Archaea which should allow rapid insight into the relative dynamics of the different members of dechlorinating communities as a response to environmental changes. Overall, CARD-FISH proved to be a rapid, reliable and convenient method to detect and quantify Dehalococcoides cells.     

A protocol for PCR in situ hybridization of hyphomycetes.

A protocol for application of Polymerase Chain Reaction (PCR) in situ hybridization for the detection of hyphomycetes is presented. The experiments are exemplary carried out with strains of the genera Penicillium and Cladosporium. The small ribosomal subunit is amplified in situ by PCR using fungal specific primers. The amplicon is used as target region for a fluorescein-marked probe. The permeability of the fungal cell wall for the primers and the probe can be successfully achieved by enzymatic treatment with beta-glucanase. The protocol can be used as a basis for further development of in situ hybridization with taxon specific probes.     

Chromosomal localization of the rat Harvey-ras-1 gene (HRAS) by in situ hybridization.

The rat Harvey-ras-1 protooncogene (HRAS) has previously been assigned to rat chromosome 1. In this study we further refine its localization to region 1q41-->q42 through the use of fluorescent in situ hybridization.     

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.     

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)     

Chromosomal localization of the major histocompatibility complex of the horse (ELA) by in situ hybridization

The first gene assignment to a horse chromosome is reported for equine leucocyte antigen (ELA), the major histocompatibility complex of the horse. A cloned DNA sequence derived from a class I gene of the porcine major histocompatibility complex was used as a probe for an in situ hybridization experiment. We present the regional localization of ELA, using this sequence, to equine chromosome 20g14-q22.     

In situ DNA‐hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms

In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non‐enzymatic, hybridization chain reaction (HCR)‐based signal amplified in situ whole‐cell detection technique (in situ DNA‐HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA‐HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)‐FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA‐HCR. In summary, in situ DNA‐HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.     

Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)     

Multicolor fluorescence in situ hybridization (M-FISH) on cells from urine for the detection of bladder cancer

Bladder cancer is the fifth most common cancer in adults. Because of the high recurrence rate (up to 70%) new tumor markers for urine are necessary for monitoring patients. In this study, we investigated the value of M-FISH on cells from urine for the detection of bladder cancer. Urine samples from 141 patients suspicious of bladder cancer were analyzed in this study. Cells were isolated from urine before surgical therapy. For FISH analysis, a commercial kit (UroVysion) containing hybridization probes for chromosomes 3, 7, 9p21 and 17, was used. Twenty-five cells were analyzed in each case by two observers. A FISH result was obtained in 121 cases. Overall, sensitivity was 60% and specificity reached 82.6%. Sensitivity and specificity by cytology were 24.1% and 90.5%, respectively. Analyzing results concerning T-category, sensitivity of FISH and cytology was 36.1% and 15% in pTa, 65.2 and 25.7% in pT1, 100% and 66.7% in pT2-3 tumors, respectively. Concerning tumor grade, similar results were obtained: sensitivity was 37% and 14% in G1, 65.4% and 40% in G2, 91.7% and 50% in G3 tumors, for FISH and cytology, respectively. In conclusion, FISH on cells from urine has been shown in all studies to be highly sensitive and specific for detection of bladder cancer. Sensitivity of FISH is higher than conventional cytology and can be used in routine diagnosis additionally to conventional cytology especially in doubtful or negative cases. FISH can detect recurrence earlier than other methods like cytology, cystoscopy or biopsy histological examination.     

Regional localization of the human glutaminase (GLS) and interleukin-9 (IL9) genes by in situ hybridization.

Phosphate-activated glutaminase is found in mammalian small intestine, brain, and kidney, but not in liver. The enzyme initiates the catabolism of glutamine as the principal respiratory fuel in the small intestine, may synthesize the neurotransmitter glutamate in the brain, and functions in the kidney to help maintain systemic pH homeostasis. Interleukin-9 (IL9) is a relatively new cytokine that supports the growth of helper T-cell clones, mast cells, and megakaryoblastic leukemia cells. cDNA clones have recently been obtained for each of these genes. The human loci for phosphate-activated glutaminase (GLS) and IL9 have previously been mapped to chromosomes 2 and 5, respectively, by analysis of somatic cell hybrid DNAs. By using chromosomal in situ hybridization, we have regionally mapped GLS to 2q32----q34 and IL9 to 5q31----q35.     

Multiprobe fluorescence in situ hybridization (UroVysion) for the detection of urothelial carcinoma - FISHing for the right catch

Urinary cytology has a well-established role in the detection and monitoring of urothelial carcinoma. The main strength of cytology is the high specificity for high-grade urothelial carcinoma and carcinoma in situ, but it has a low sensitivity for low-grade, non-invasive tumors. There are several other limitations of cytology. Cytology of the upper urinary tract and after intravesical therapy with bacillus Calmette-Guerin is notoriously difficult to interpret. In addition, there is a poorly defined but commonly used category of atypical cytology of uncertain significance. The UroVysion multiprobe fluorescence in situ hybridization has emerged as a helpful tool to address these limitations. It consists of fluorescently labeled DNA probes to detect increased copy numbers (polysomy) of the chromosomes 3, 7 and 17 and deletion of 9p21, the site of the P16 tumor suppressor gene. Multiple studies have shown that fluorescence in situ hybridization in voided urine and washing specimens can help in patient management due to its superior sensitivity over cytology in different situations. It can be particularly useful to clarify equivocal cytological findings. However, some aspects remain to be further addressed including cost efficiency, optimal cut-off values and the true performance under real-life conditions.     

Application of PCR in situ method for detection of human cytomegalovirus (HCMV) DNA.

Present HCMV diagnosis is relatively slow and inefficient. We applied PCR in situ to 15 samples of human salivary glands, 20 cytospins from blood, and 10 human fibroblast samples in order to detect the presence of HCMV DNA. The results indicate that PCR in situ is an effective and very sensitive method for detection of HCMV infections in variety of specimens.     

Specific detection of Arcobacter and Campylobacter strains in water and sewage by PCR and fluorescent in situ hybridization

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10(3) cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.