Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization

Oligonucleotide probes specific to Cryptosporidium parvum (CRY1) were conjugated with a range of fluorochromes. The fluorescence after in situ hybridization (FISH) labelling of oocysts and controls was assessed. The objective was to determine the most suitable conjugate for FISH labelling, followed by analysis with a 488 nm laser flow cytometer. The most promising candidate was fluorescein isothiocyanate but only when linked to the CRY1 probe via an 18-carbon spacer arm consisting of six ethylene glycol moieties. The use of the spacer increased fluorescent signals fivefold compared with an equivalent probe in which the FITC was linked directly to the 5'-amino group of the DNA.     

Flow cytometric detection of saxitoxins using fluorescent voltage-sensitive dyes

By virtue of their ability to block depolarization of nerve cells, the saxitoxins exert the toxic effects associated with paralytic shellfish poisoning and allow for their detection through various methodologies. When veratridine-induced depolarization is followed using voltage-sensitive fluorescent dyes, the presence of these toxic blocking agents can be observed as a decrease in fluorescence of dye-treated nerve cells. Detection using flow cytometry provides for selection of the most responsive population of cultured mouse neuroblastoma (Neuro 2a) cells thereby enhancing assay sensitivity and this approach can be accomplished in real time. The method is demonstrated in preliminary studies using saxitoxin and crude shellfish extracts.     

Specific detection of RNA molecules by fluorescent in situ hybridization in living cells

The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.Abbreviations PI propidium iodide - SLO streptolysin O     

Oligonucleotide probes for specific detection of Giardia lamblia cysts by fluorescent in situ hybridization

AIMS: Our study focused on the design of oligonucleotide probes and a suitable hybridization protocol that would allow rapid and specific identification of potentially viable cysts of the waterborne parasite Giardia lamblia. METHODS AND RESULTS: Comparative analysis of ribosomal RNA (rRNA) sequences of Giardia lamblia and a number of closely and more distantly related species identified six regions that appear to be specific for the G. lamblia 16S rRNA. Fluorescently labelled probes targeting these regions were produced and employed in fluorescent in situ hybridization (FISH) experiments. Two of the six probes tested successfully. CONCLUSION: Our study provides the first reported probes for specific FISH detection of G. lamblia. The method depends on sufficient amounts of intact rRNA in the target organism, which is unlikely to be present in nonviable cysts that have been exposed to the environment for a prolonged period. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, detection of G. lamblia cysts is largely based on immunofluorescence assays (IFA) targeting cyst wall surface antigens. These assays lack specificity and will detect species others than G. lamblia. Further, IFA will detect nonviable cysts and cyst wall fragments that do not pose a public health risk. In contrast, FISH probes allow specific detection and are likely to only detect viable, infectious cysts.     

Routine diagnosis of DiGeorge syndrome by fluorescent in situ hybridization

In a series of ten patients affected by DiGeorge syndrome, we screened, by high resolution banding and fluorescent in situ hybridization of a cosmid probe, for microdeletions associated with this syndrome. In the ten patients, a microdeletion was demonstrated by in situ hybridization, but suspected only in two patients by high resolution banding.     

Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification.

To understand the biological relationships among various molecules, it is necessary to define the cellular expression patterns of multiple genes and gene products. Relatively simple methods for performing multi-label immunohistochemical detection are available. However, there is a paucity of techniques for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection systems and simplified ISH protocols has prompted us to develop a tyramide signal amplification method for sequential multi-label fluorescent ISH and IHC detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neurotrophic factor receptor alpha2 (GFRalpha2) mRNA expression and IHC localization of its co-receptor Ret in the trigeminal ganglion of postnatal Day 0 mice. We found that approximately 70% of Ret-immunoreactive neurons possessed GFRalpha2 mRNA and virtually all GFRalpha2-expressing neurons contained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed, paraffin-embedded sections and a monoclonal antibody against neuron-specific nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFRalpha2 mRNA expression in adult mouse brain. This multi-labeling technique should be applicable to a wide variety of tissues, antibodies, and probes, providing a relatively rapid and simple means to compare mRNA and protein localization.     

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.     

Detection of the oyster parasite Bonamia ostreae by fluorescent in situ hybridization

Bonamia ostreae is an economically significant protistan parasite of the flat oyster Ostrea edulis in Europe and North America. Management of this parasite depends partly upon its reliable identification in wild and aquacultured oyster populations, but B. ostreae is small and difficult to detect by traditional microscopic methods. We designed a fluorescent in situ hybridization (FISH) assay to sensitively detect B. ostreae in standard histopathological sections of B. ostreae-infected oysters using fluorescently labeled DNA oligonucleotide probes. Hybridization using a cocktail of 3 presumptively B. ostreae-specific, fluorescein iso(thio)cyanate (FITC)-labeled oligonucleotides produced an unambiguous staining pattern of small green rings inside infected oyster hemocytes that was easily distinguished from host tissue background. This pattern is diagnostic for B. ostreae. A negative control cocktail of oligonucleotides containing 2 mismatches relative to target sequences, on the other hand, failed to hybridize at all. B. ostreae-specific probes did not cross-react with a related protist, Haplosporidium nelsoni.     

Single copies of HIV proviral DNA detected by fluorescent in situ hybridization

A fluorescent in situ DNA hybridization assay employing a biotinylated DNA probe was used to visualize single copies of human immunodeficiency virus (HIV) proviral DNA in the nuclei and metaphase chromosomes of infected cells. In clonal cell lines that contain either one or two copies of proviral DNA, the efficiency of detection of individual loci of proviral DNA was 57% to 78%. Only 1% of uninfected control cells exhibited a false-positive signal. HIV proviral DNA could be accurately identified in mixed populations comprised of only 5% infected cells. Thus, this assay could be used to identify cells that harbor HIV proviral DNA and to monitor the status of proviral DNA throughout the course of HIV infection.     

AcroM fluorescent in situ hybridization analyses of marker chromosomes

The presence of a de novo supernumerary marker chromosome (SMC) poses problems in genetic counseling. The consequences of the additional chromosomal material may range from harmless to detrimental. As the composition of a SMC cannot be deciphered by traditional banding analysis, sophisticated methods are needed for their rapid and detailed analyses. A new strategy is presented, which allows the elucidation of the composition of SMCs in one or two hybridizations. One hybridization, termed AcroM-FISH, involves a newly generated probe mix, which consists of painting probes for all acrocentric chromosomes, centromere probes for chromosomes 13/21, 14/22, 15, and a probe specific for rDNA, each labeled with a specific combination of fluorochromes. This probe mix is sufficient to characterize approximately 80% of all SMCs. For the other 20% of SMCs, chromosomes can be analyzed in a second hybridization by multicolor karyotyping, for example, multiplex FISH (M-FISH), to check for the presence of euchromatin of other chromosomes. The potential of AcroM-FISH was tested in various applications.     

Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction

Abstract Mycelium of Pleurotus ostreatus var. florida with a decreased growth rate contained seven double-stranded RNA segments and isometrical virus particles with diameters of 24 and 30 nm. Mycelium with a normal growth rate lacked dsRNA. Protoclones from virus-containing mycelium contained one to seven of these dsRNA segments in varying concentrations. The exact correlation between slow growth and the presence of dsRNA molecules could not be established. Infection of virus-free protoplasts with PEG-precipitated virus particles resulted in mycelium that stably maintained the 2.4 kbp dsRNA.