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1.
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.  相似文献   

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Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination, cellular identification and gene expression, monitoring viral multiplication in infected cells, and bacterial community analysis and enumeration. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T(m)) of these analogs for natural nucleic acids. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes and have been successfully used to detect eukaryotic mRNA and viral RNA in mammalian cells. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).  相似文献   

4.
Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.  相似文献   

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Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.  相似文献   

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During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.  相似文献   

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In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.  相似文献   

9.
In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant–fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period.  相似文献   

10.
Although fluorescence in situ hybridization (FISH) with specific ribosomal RNA (rRNA)‐targeted oligonucleotides is a standard method to detect and identify microorganisms, the specific detection of genes in bacteria and archaea, for example by using geneFISH, requires complicated and lengthy (> 30 h) procedures. Here we report a much improved protocol, direct‐geneFISH, which allows specific gene and rRNA detection within less than 6 h. For direct‐geneFISH, catalyzed amplification reporter deposition (CARD) steps are removed and fluorochrome‐labelled polynucleotide gene probes and rRNA‐targeted oligonucleotide probes are hybridized simultaneously. The protocol allows quantification of gene copy numbers per cell and the signal of the directly labelled probes enables a subcellular localization of the rRNA and target gene. The detection efficiencies of direct‐geneFISH were first evaluated on Escherichia coli carrying the target gene on a copy‐control vector. We could show that gene copy numbers correlated to the geneFISH signal within the cells. The new protocol was then applied for the detection of the sulfate thiolhydrolase (soxB) genes in cells of the gammaproteobacterial clade SUP05 in Lake Rogoznica, Croatia. Cell and gene detection efficiencies by direct‐geneFISH were statistically identical to those obtained with the original geneFISH, demonstrating the suitability of the simpler and faster protocol for environmental samples.  相似文献   

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Conventional, morphological identification of ciliates and other protozoa needs considerable experience and often is difficult as various staining methods must be applied. New molecular techniques, such as fluorescence in situ hybridization (FISH) with gene probes, are powerful means to overcome this problem. As a test case, the morphology of two very similar, and thus difficult to differentiate ciliate morphospecies, Glaucoma scintillans and Glaucomides bromelicola, were compared. They were then distinguished by applying the Ciliate-FISH technique with a set of eight 18S rRNA targeted oligonucleotide probes, four of which have been developed for specific detection of G. scintillans. The remaining four probes were designed to detect G. bromelicola in order to prove probe specificities by binding to the homologous target region of the probes mentioned before. The tests resulted in a clear and easy differentiation of the two species by fluorescence signals of three of the four tested probe pairs. Thus, FISH techniques are very useful for the identification and detection of protozoa and might be of great help studying geographical distributions of known taxa.  相似文献   

12.
RNA dependence in the cell cycle of V79 cells   总被引:1,自引:0,他引:1  
The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.  相似文献   

13.
Almost one thousand 16S rRNA sequences of Gram-positive bacteria with a low DNA G + C content from public databases were analyzed using the ARB software package. A signature region was identified between positions 354 and 371 (E. coli numbering) for the Bacillus sub-branch of the Gram-positive bacteria with a low DNA G + C content, the former orders Bacillales and Lactobacillales. Three oligonucleotide probes, namely LGC354A, LGC354B, and LGC354C, were designed to target this diagnostic site. Their fluorescent derivatives were suitable for whole cell detection by fluorescence in situ hybridization (FISH). Hybridization conditions were adjusted for differentiation of target and related non-target reference species. When applying FISH to whole bacterial cells in a sample of activated sludge from a communal wastewater treatment plant, members of the Bacillus sub-branch were detected at levels from 0.01% of cells in samples fixed with paraformaldehyde to over 8 percent in the same samples fixed with ethanol and treated with lysozyme. The problems of quantitative in situ analysis of Gram-positive bacteria with a low DNA G + C content in biofilm flocs are discussed and recommendations made. Members of the Bacillus sub-branch were detected in different abundances in activated sludge samples from different wastewater plants.  相似文献   

14.
Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).  相似文献   

15.
Fluorescent in situ hybridisation (FISH) with a 16S ribosomal RNA (rRNA)-targeted oligonucleotide probe, Eub338, could be used to estimate the in situ activity of Lactobacillus plantarum WCFS1 in exponentially growing cells. However, L. plantarum is capable of growth to very high cell densities, and the properties of the L. plantarum cell envelope prevented effective entry of the fluorescent oligonucleotide probe into the cells at later stages of growth at high cell densities. Total rRNA measurements of cells isolated at different growth stages showed maximal amounts of RNA (8.77+/-0.8 fg) per cell at the early stationary phase and confirmed the effectiveness of FISH for accurate activity measurement in exponentially growing cells.  相似文献   

16.
A capillary electrophoresis (CE)-based technique is reported here to monitor differential RNA synthesis in individual Chinese hamster ovary cells at distinct stages of the cell proliferation cycle. Cell synchronization was achieved by the shake-off method, in which mitotic (M) cells were dislodged, and cells at G(1), S, and G(2) phases were harvested 2.5, 10, and 13 h, respectively, after synchronizing the mitotic cells. Thirty-two cells (eight from each phase) were analyzed by injecting each cell into the capillary, lysing it with dilute surfactant, separating the RNA by capillary electrophoresis, and detecting the peaks with laser-induced fluorescence. The results from single cells show that the total amount of RNA increased at each successive stage (from G(1) to M), while the relative synthetic rates of different RNA fractions varied with progression through the cycle. There was a threefold increase in the synthetic rate of total RNA from S to G(2), compared with G(1) to S. In addition, differential accumulation of specific RNA fractions was observed, with the low-molecular-mass fraction exhibiting a much higher synthetic rate from G(2) to M, relative to the rates of the larger ribosomal RNA (rRNA) fractions. Comparison of the large rRNA fractions with one another reveals that at S phase more 28S rRNA was accumulated than 18S rRNA, and at G(1) and M phases, the synthetic rate of 28S rRNA was slowed compared with that of 18S. Minimal sample preparation, combined with the separation power of CE and single-cell detection sensitivity of laser-induced fluorescence, results in a simple method for assessing differential accumulation of RNA from distinct individual cells.  相似文献   

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We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.  相似文献   

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Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO-FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO-FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO-FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO-FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution.  相似文献   

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