Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model

Introduction

In this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting the C57BL/6 mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary to a sequence of the H. pylori 16S rRNA gene, was used. First, the potential cytotoxicity and genotoxicity of the probe was assessed by commercial assays. Further, the performance of the probe for detecting H. pylori at different pH conditions was tested in vitro, using fluorescence in situ hybridization (FISH). Finally, the efficiency of FIVH to detect H. pylori SS1 strain in C57BL/6 infected mice was evaluated ex vivo in mucus samples, in cryosections and paraffin-embedded sections by epifluorescence and confocal microscopy.

Results

H. pylori SS1 strain infecting C57BL/6 mice was successfully detected by the Cy3_HP_LNA/2OMe_PS probe in the mucus, attached to gastric epithelial cells and colonizing the gastric pits. The specificity of the probe for H. pylori was confirmed by microscopy.

Conclusions

In the future this methodology can be used in combination with a confocal laser endomicroscope for in vivo diagnosis of H. pylori infection using fluorescent LNA probes, which would be helpful to obtain an immediate diagnosis. Our results proved for the first time that FIVH method is applicable inside the body of a higher-order animal.     

Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2’-O-methyl RNAs (2’OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others.     

Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.     

In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes

New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes.     

Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification

Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).     

Specific Detection of Dehalococcoides Species by Fluorescence In Situ Hybridization with 16S rRNA-Targeted Oligonucleotide Probes

Dehalococcoides ethenogenes is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of Dehalococcoides species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for Dehalococcoides species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except D. ethenogenes and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of Dehalococcoides species. In situ hybridization of probe Dhe1259t with D. ethenogenes strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active Dehalococcoides species in these enrichment samples.     

Detection of Bacteria in Phagocyte-Smears from Septicemia-Suspected Blood by In Situ Hybridization Using Biotinylated Probes

We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization. This was obtained by using nick-translated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia.     

Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.     

Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility

Fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5′-, 3′-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase in situ accessibility of rRNA target sites and thus provide more flexibility for probe design.Since its introduction almost 20 years ago (2, 7), the identification of microorganisms by fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted probes has found widespread application in environmental and medical microbiology (1, 16). Despite being a methodologically robust technique, standard FISH suffers from several limitations (26) that may prevent successful detection of target microorganisms. One of the most frequently reported FISH problems is a low signal intensity of the detected microbes.Dim signals can be caused by a low cellular concentration of the target molecules (16S rRNA or 23S rRNA), a feature typically found in microorganisms thriving in oligotrophic environments (19). In order to increase the sensitivity of FISH and make it suitable for the detection of microbes with a low ribosome content, several strategies have been developed (6, 13, 18, 19, 21, 25), of which catalyzed reporter deposition (CARD)-FISH has found the most widespread application. The CARD-FISH technique (18, 21) uses horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification and achieves a 26- to 41-fold-higher sensitivity than standard FISH (11). However, CARD-FISH is rather expensive, requires enzymatic pretreatment to allow the large horseradish peroxidase-labeled probes to penetrate the target cells (17), and causes a dramatically altered melting behavior of the probes (11).Another frequently encountered FISH problem is the low in situ accessibility of many regions of the 16S and 23S rRNA for singly labeled probes (9, 10). Probes targeting such regions, which comprise about one-third of the Escherichia coli 16S rRNA (10), confer signals which are very dim or even below the detection limit. In order to avoid the selection of poorly accessible target sites for FISH probe design, a consensus 16S rRNA accessibility map for prokaryotes has been established based on detailed accessibility maps of two bacterial model organisms and one archaeal model organism (3). Considering this consensus map during probe design is recommended, but it excludes many probes with useful specificities from FISH applications. Furthermore, accessibility of many 16S and 23S rRNA target sites varies between different microorganisms and thus cannot yet be reliably predicted in silico. Accessibility of target sites to probes can be improved by the following: (i) use of unlabeled helper probes (8), (ii) elongation of the hybridization time up to 96 h (30), (iii) elongation of the probes, resulting in an altered ΔG°_overall (30), or (iv) use of peptide nucleic acid probes (reference 26 and references therein). However, all these strategies have specific limitations. For example, the design of helper probes is often impossible for probes with broader specificities, the extension of the hybridization time might lead to unspecific probe or dye binding in complex samples, probe elongation is often not possible without narrowing its specificity, and previously published oligonucleotide probes cannot simply be converted into the expensive peptide nucleic acid probes without a dramatically changed specificity (26).In principle, using oligonucleotide probes labeled with multiple fluorescent dyes should provide a simple means to increase the FISH signal intensity. The first experiments with multilabeled oligonucleotides were performed briefly after the introduction of the technique in microbiology but resulted in a pronounced increase in unspecific staining of nontarget organisms and/or an unexpected decrease in the signal intensity of the target organism which was attributed to quenching effects (28). Inconsistent with these data, Spear et al. (23) reported a successful increase in the signal-to-noise ratio of FISH-detected fungal cells by application of a multilabeled 18S rRNA-targeted oligonucleotide probe. In this work, we systematically evaluated the effect of 5′- and 3′-doubly labeled oligonucleotide probes (which were not included in the previously published study of multilabeled probes [28]) on the FISH signal intensity of Gram-negative and Gram-positive cells and studied the influence of double labeling on the in situ accessibility of rRNA target sites.The double-labeling-of-oligonucleotide-probes (DOPE)-FISH approach was initially tested with four bacterial pure cultures. These included the gamma- and betaproteobacterial Gram-negative species Escherichia coli (DSM 498) and Burkholderia cepacia (DSM 7288), respectively. In addition, the two Gram-positive bacteria Bacillus subtilis (DSM 10) and Listeria monocytogenes (strain LO28) were used. E. coli, B. cepacia, and B. subtilis were grown according to the DSMZ instructions until they reached their stationary growth phase and were fixed with paraformaldehyde (E. coli and B. cepacia) or ethanol (B. subtilis) as described elsewhere (5). L. monocytogenes strain LO28 was grown on brain heart infusion for 5 h and fixed with ethanol as outlined previously (27). The oligonucleotide probes EUB338 (targeting the 16S rRNA of most but not all bacteria), NonEUB338 (a nonsense probe), GAM42a (targeting the 23S rRNA of many members of the Gammaproteobacteria), and five E. coli-targeted probes with a low 16S rRNA accessibility (10) were obtained as singly and doubly labeled derivatives from Thermo Hybaid (Interactiva Division, Ulm, Germany). More information about the applied probes can be found at probeBase (14) and in the publication by Fuchs et al. (10). FISH was performed by following the standard protocol (5) under the conditions recommended for each probe (14). If not stated otherwise, all hybridizations were carried out with identical hybridization (4 h) and washing times (10 min), respectively. Probe-conferred signal intensities were quantified using a confocal laser scanning microscope (CLSM) (LSM 510 Meta; Zeiss, Oberkochen, Germany) and the software program daime (4) by analyzing at least 1,000 single cells per experiment. For these measurements, individual cells were detected by image segmentation via edge detection.Regardless of the dye used (Cy3, Cy5, or FLUOS) and the respective target organism analyzed, hybridization with the doubly labeled probe EUB338 resulted in an increase in the FISH signal compared to the use of the singly labeled probe EUB338. For three of the four reference organisms, this increase was about 2-fold, while an even stronger signal amplification was observed for B. cepacia (Fig. ​(Fig.1A).1A). Consequently, the distance between the two dye molecules in 18-nucleotide probes labeled at both ends is sufficient to avoid quenching. This is in contrast to oligonucleotides which are multiply labeled at one end or within the probe (28). Hybridization of the four reference organisms with the singly and doubly labeled derivatives of probe GAM42a confirmed these results and demonstrated that double labeling does not increase the background fluorescence of nontarget microorganisms (Fig. ​(Fig.1B).1B). Consistent with these findings, hybridization of all reference organisms with a doubly labeled nonsense probe (with FLUOS, Cy3, and Cy5) resulted in signals below the detection limit of the CLSM if standard FISH settings were applied (data not shown). We also evaluated the influence of the hybridization time on the signal intensity achievable by DOPE-FISH by varying the hybridization time between 1 and 6 h. In these experiments, no significant difference in DOPE-FISH signal intensities of the Gram-positive and Gram-negative reference organisms were observed, indicating that the additional label of the DOPE-FISH probes does not dramatically influence the hybridization kinetics (data not shown).Open in a separate windowFIG. 1.(A) Effect of double labeling of the EUB338 probe on the FISH signal intensity of four reference organisms. For each organism, the signal intensity conferred by a doubly labeled EUB338 probe was normalized to the signal intensity obtained with the same probe as a singly labeled derivative. Hatched, light-gray, and dark-gray bars depict results with the Cy3-, Cy5- and FLUOS-labeled probe EUB338, respectively. (B) Effect of double labeling of the probe Gam42a (in the presence of the unlabeled competitor probe Bet42a, specific for most members of the Betaproteobacteria [14]) on the FISH signal intensities of four reference organisms. For each organism, the signal intensity conferred by the doubly labeled probe Gam42a was normalized to the signal intensity obtained for E. coli with the same probe as a singly labeled derivative. Hatched, light-gray, and dark-gray bars depict results with the Cy3-, Cy5-, and FLUOS-labeled probe, respectively. The weak unspecific signals observed with some DOPE-FISH probes for B. subtilis are also detectable at comparable intensities with singly labeled probes (data not shown). (C) Cy3-doubly labeled but not FLUOS-doubly labeled probes improve in situ accessibility of E. coli 16S rRNA target sites. E. coli was hybridized with five probes representing brightness classes V and VI (3). FISH signals were recorded for Cy3-singly and -doubly labeled probes and normalized to the FISH signal obtained for E. coli with the singly labeled probe EUB338. Light-gray and dark-gray bars depict results with Cy3-singly and doubly labeled probes, respectively. FLUOS-singly and -doubly labeled probes showed no signal. For all panels, all experiments were performed in triplicate. Error bars indicate the standard deviation. ND, not detectable.Although DOPE-FISH worked well with all tested reference organisms, it should be noted that the Gram-positive species L. monocytogenes showed a signal amplification only if it was treated with lysozyme (according to Wagner et al. [27]) prior to the application of the doubly labeled probe. Without this enzymatic pretreatment, application of the singly labeled probe EUB338 resulted in a stronger signal than was seen with the doubly labeled probe EUB338, indicating that for some Gram-positive microorganisms with dense cell walls, double labeling impairs probe penetration. The observation that lysozyme treatment of L. monocytogenes also enhanced the probe-conferred signal of the singly labeled EUB338 probe confirmed that the cell wall of this organism after ethanol fixation is also not freely permeable to singly labeled probes (27; also data not shown). Enzymatic pretreatment of fixed microbial cells is also routinely applied for successful application of CARD-FISH, but since this method uses peroxidase-labeled probes which are much larger than DOPE-FISH probes, such treatments are also used for Gram-negative bacteria (11). Although many microorganisms are detectable by CARD-FISH with enhanced signal intensities, appropriate pretreatment protocols are not yet available for all microbes. For example, the sheathed filamentous methane oxidizer Crenothrix polyspora can easily be detected by standard FISH (24), but only very few cells within the filaments show a signal after CARD-FISH even if harsh permeabilization pretreatments are applied (see Fig. S1A in the supplemental material), a phenomenon known for sheathed microorganisms (12). In contrast, detection of all Crenothrix cells with more than 2-fold-increased signal intensity is readily possible by DOPE-FISH (see Fig. S1B).Prior research has demonstrated that probe labeling with horseradish peroxidase for CARD-FISH dramatically alters the melting behavior of oligonucleotide probes (11). Therefore, we recorded melting curves for Cy3-, Cy5-, and FLUOS-singly and -doubly labeled probes EUB338 and Gam42a by applying increasingly stringent conditions in the hybridization and wash steps (5). Interestingly, these experiments showed that the FLUOS singly labeled probes formed less-stable duplexes with their target sequences than the respective Cy3- and Cy5-labeled probes (Fig. ​(Fig.2).2). This effect, which is consistent with recent data on the stabilizing effect of various fluorophores on model probe-target duplexes (15), indicates that FLUOS-labeled FISH probes are generally applied under more-stringent conditions than Cy3- or Cy5-labeled probes. Cy3- and Cy5-doubly labeled probes displayed with their target organisms probe dissociation profiles similar to those of the respective FLUOS singly labeled probes, demonstrating that Cy3 or Cy5 double labeling does not further stabilize but rather moderately weakens the probe-target hybrid. Consistent with these findings, doubly FLUOS-labeled probes showed the lowest T_m (Fig. ​(Fig.2).2). Importantly, double labeling of probe GAM42a did not adversely affect mismatch discrimination, as shown by its dissociation profiles if in situ hybridizations were performed at various stringencies with B. cepacia containing a single mismatch in the probe target site of its 23S rRNA. (Fig. ​(Fig.2B).2B). These results indicate that the specificities of DOPE-FISH probes can be regarded as identical to those of standard singly labeled FISH probes.Open in a separate windowFIG. 2.Comparison of probe dissociation profiles of singly and doubly labeled probes. For each profile, the microscopic settings were adjusted for the lowest formamide concentration and subsequently kept constant. Dashed and solid lines represent sigmoid fittings for singly and doubly labeled probes, respectively. (A) Dissociation profiles of the singly and doubly labeled probe Gam42a with E. coli as the target organism. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe GAM42a, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe GAM42a, respectively. (B) Dissociation profiles of the singly and doubly labeled probe Gam42a with B. cepacia as a nontarget organism having a single mismatch to probe GAM42a. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe GAM42a, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe GAM42a, respectively. The melting curves for FLUOS-singly labeled and Cy5-doubly labeled probes are almost identical and thus overlap. In the presence of the unlabeled probe Bet42a as a competitor, no probe-conferred signal was recordable for both singly and doubly labeled GAM42a probes. (C) Dissociation profiles of the singly and doubly labeled probe EUB338 with E. coli as the target organism. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe EUB338, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe EUB338, respectively. For all panels, error bars are not shown since they were always smaller than the symbols.In order to analyze whether the in situ accessibility of rRNA target sites to doubly labeled probes differs from that to those labeled with only one dye, we tested five probes targeting E. coli 16S rRNA. These probes were described as yielding only very dim signals with standard FISH as a consequence of limited target site accessibility and were thus assigned to the lowest brightness classes, V or VI (3, 10). Consistently, standard FISH with these five singly labeled probes (Cy3 and FLUOS) gave no or very weak signals (Fig. ​(Fig.1C).1C). Unexpectedly, however, Cy3-doubly labeled derivatives of these probes produced much brighter signals (Fig. ​(Fig.1C),1C), and for some of the probes (Eco468 and Eco1310), the DOPE-FISH signal intensity was higher than that measured for the Cy3-singly labeled probe EUB338 (Fig. ​(Fig.1C).1C). One could speculate that a Cy3 label at the 3′ end and not the double labeling might be responsible for the improved accessibility of rRNA target sites for Cy3 DOPE-FISH probes. However, since selected probes (Eco262, Eco468, and Eco1310) labeled with a single Cy3 molecule at the 3′ end did not result in increased fluorescence, this hypothesis can be rejected (data not shown). Interestingly, FLUOS-labeled DOPE-FISH probes did not show increased fluorescence, strongly indicating that the improved accessibility of Cy3 DOPE-FISH probes depends on the chemical structure of the fluorophore. This is consistent with the observation that Cy5 double labeling of the five E. coli probes also resulted in improved probe accessibilities (data not shown). While Cy3 and Cy5 double labeling decreases the probe-target duplex stability (Fig. ​(Fig.2),2), it apparently helps to resolve secondary or tertiary structures responsible for poor in situ accessibility of rRNA target sites. It is tempting to speculate that binding of Cy3 or Cy5 to double-stranded rRNA regions, analogous to the previously reported intercalation of certain cyanine class dyes in DNA (29) or other modes of nucleic acid binding by cyanine dyes (15), contributes to this phenomenon.The improved accessibility of rRNA target sites for Cy3 DOPE-FISH probes offers more flexibility for probe design because it enables the use of probes with excellent specificity but low standard FISH signal intensity for the successful in situ detection of microbes. This advantage of DOPE-FISH is nicely demonstrated by the probe Ntspa175 (5′-GAC CAG GAG CCG TAT GCG-3′), which targets the 16S rRNA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU229885","term_id":"281398158","term_text":"GU229885"}}GU229885) of an uncultured nitrite oxidizer of the genus Nitrospira thriving in activated sludge. At 25% formamide in the standard FISH hybridization buffer (5), this probe is highly specific as demonstrated by Clone-FISH (22) using another activated sludge-derived 16S rRNA Nitrospira-like sequence with a single mismatch to probe Ntspa175 as a nontarget control (data not shown). Standard FISH of activated sludge with the Cy3-labeled probe Ntsp175, which targets the 175-to-193 region in the 16S rRNA, resulted in the detection of Nitrospira microcolonies with very variable FISH signal intensities. A considerable number of stained microcolonies had extremely dim FISH signals, indicating that these cells had a ribosome content too low to be reliably detectable by a standard FISH probe of a low brightness class. DOPE-FISH of the same sample with the Cy3-doubly labeled probe Ntspa175 led to a pronounced increase in signal intensity of the target cells (see Fig. S2 in the supplemental material) without causing increased background fluorescence if standard confocal-microscope settings were applied. In accordance with this observation, the relative biovolume-abundance of the detectable Ntspa175-stained population compared to the biovolume of those cells labeled by the Nitrospira genus-specific probe Ntspa662 (14) in the activated sludge increased by a factor of 1.81 ± 0.1 if a doubly labeled Ntspa175 probe was used (measurements made by the software package daime using confocal-microscope images as described previously [4]).In summary, DOPE-FISH with commercially available doubly labeled oligonucleotide probes is a straightforward modification of the standard FISH procedure which increases the signal intensity of standard FISH probes by at least a factor of 2 without causing specificity problems. Importantly, the influence of DOPE-FISH on the dissociation profile of probes is not larger than that caused by a dye switch from Cy3 to FLUOS if singly labeled probes are used for FISH. Thus, previously optimized hybridization and washing conditions for published probes can be applied for DOPE-FISH. Since DOPE-FISH unlocks previously inaccessible target sites on the rRNA, this new FISH approach offers more options for the design of specific probes, a task which becomes increasingly difficult with the rapid growth of rRNA databases (20).      

荧光原位杂交技术在医学微生物学中的应用

以16S rRNA 为靶序列的寡核苷酸探针荧光原位杂交技术已广泛应用于分析复杂环境中的微生物群落构成,包括监测和鉴定病原微生物以及未被培养微生物．通过对临床样品中微生物细胞的检测能提供微生物在人体中的种类、数量和空间分布等信息．其结果快速准确,较之传统的病原微生物诊断方法具有明显的优越性,在临床应用中有广泛的前景．     

On-Line Detection of Substrate Exhaustion by Using NAD(P)H Fluorescence

In this study we examined the utility of NAD(P)H fluorescence for monitoring aerobic fermentations of the threonine auxotroph Corynebacterium glutamicum ATCC 14296. Instead of attempting complicated mathematical corrections for inner-filter effects, we found that it is possible to use the information contained in the on-line NAD(P)H fluorescence signal to assess culture metabolic activities during fermentation. The first derivative of the filtered fluorescence signal, which approximates the turnover rate of the NAD(P)H pool, can be used to precisely identify the temporal points of threonine and glucose exhaustion.     

Detection of Staphylococci in Mouse Phagocytic Cells by in Situ Hybridization Using Biotinylated Dna Probes

In situ hybridization was used to detect intracellular Staphylococcus aureus and S. epidermidis in mouse phagocytic cells after experimental infection of C3H mice with Staphylococci via abdominal or intravenous injection. Isolated ascites or whole blood were tested by the phagocyte smear technique, using bacteriolytic enzymes to preserve phagocytic cell morphology. The exposed bacterial DNA was visualized as intracellular hybridized signals by use of biotinylated DNA probes and by immunocytochemistry using streptavidin-alkaline phosphatase conjugates as detector molecules. These DNA probes, prepared from randomly cloned genomic DNA fragments of S. aureus and S. epidermidis, were strain-specific and did not cross-hybridize either in situ or on dot-blot hybridization. This technique of in situ hybridization with phagocyte smears is useful for detection and diagnosis of intracellular bacteria regardless of viability.     

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

Background

Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.

Methods and Results

Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.

Conclusion

Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.     

Detection of Neuropeptide mRNA in Rat Brain by In Situ Hybridization with Radiolabeled Synthetic Oligonucleotide Probes

Abstract

Radiolabeled synthetic oligonucleotide probes were used for detection of somatostatin and vasopressin mRNA in rat brain by in situ hybridization.     

Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

Malaria is a responsible for approximately 600 thousand deaths worldwide every year. Appropriate and timely treatment of malaria can prevent deaths but is dependent on accurate and rapid diagnosis of the infection. Currently, microscopic examination of the Giemsa stained blood smears is the method of choice for diagnosing malaria. Although it has limited sensitivity and specificity in field conditions, it still remains the gold standard for the diagnosis of malaria. Here, we report the development of a fluorescence in situ hybridization (FISH) based method for detecting malaria infection in blood smears and describe the use of an LED light source that makes the method suitable for use in resource-limited malaria endemic countries. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probe that detects all five species of Plasmodium known to cause the disease in humans. The P. falciparum (PF) FISH assay and P. vivax (PV) FISH assay detect and differentiate between P. falciparum and P. vivax respectively from other Plasmodium species. The FISH assays are more sensitive than Giemsa. The sensitivities of P-Genus, PF and PV FISH assays were found to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of Plasmodium, P. falciparum and P. vivax by Giemsa staining respectively.     

In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.     

用万能引物PCR法从YAC中分离制备荧光原位杂交探针的研究

报道了一种从微量且未知碱基顺序的YAC DNA中制备FISH探针的新方法——万能引物PCR(UP PCR),即把复性后的UP-Linker连接于PFGE分离后经Alu Ⅰ酶切的YAC DNA上,再用UP对其进行PCR扩增和标记。由于Alu Ⅰ的广谱性和UP与Linker长度不同等,使该法能根据PCR效率调节扩增产物的广泛性向特异性的转变,且产物能覆盖YAC嵌入DNA的全长。此法制备的人21号染色体FISH探针,特异性强,杂交信号明亮,21号染色体的检出率高。该法稳定简便。     

Rapid Discrimination of Haemophilus influenzae,H. parainfluenzae,and H. haemolyticus by Fluorescence In Situ Hybridization (FISH) and Two Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Platforms

Background

Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level.

Methodology

A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction.

Principal Findings

Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system''s database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients.