An Enzymic Method of Preparing Plant Chromosomes for In Situ Hybridization

A method of preparing chromosomes from plant root tips for in situ hybridization with tritiated DNA is described. The technique relies on the enzymic hydrolysis of plant cell walls with a pectinase-cellulase mixture. It is shown that, despite the enzymic mixture possessing nuclease activity, there is no detectable degradation of DNA within fixed root tips. To demonstrate the suitability of this method of preparing plant chromosomes for in situ hybridization, a cloned repetitive DNA sequence has been hybridized to Allium sativum chromosomes. Chromosomes prepared using this technique also can be readily G-banded.     

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.     

In Situ Hybridization of Biotinylated Dna Probes to Cotton Meiotic Chromosomes

A modified procedure for in situ hybridization of biotinylated probes to meiotic chromosomes of cotton has been developed with high retention of squashed cells on slides, preservation of acid-fixed chromosome morphology, exceptionally low levels of background precipitate at nonspecific hybridization sites and improved photomicrographic recording. Salient features of the techniques include pretreatment of slides before squashing, cold storage of squash preparations, and use of interference Biters for distinguishing precipitate from chromatin. A cloned 18S/28S ribosomal DNA fragment from soybean was biotinylated via nick-translation and hybridized to microsporocyte meiotic chromosomes 6f cotton (Gostypium hirsutum L. and G. hirsutum L. X G. barbadense L.). Enzymatically formed precipitate from streptavidin-bound peroxidase marked the in situ hybridization.

In situ hybridization of biotinylated probes to cotton meiotic chromosomes adds the specificity and resolution of in situ hybridization to the chromosomal and genomic perspectives provided by meiotic cytogenetic analyses. Molecular cytogenetic analyses of meiotic cells offer certain inherent analytical advantages over analyses of somatic cells, e.g., in terms of mapping, and for studying fundamental biological and genetic problems, particularly for organisms that are not amenable to somatic karyotypic analysis.     

In Situ Hybridization of Plant Meiotic and Mitotic Chromosomes: Differences in Signal Detection

The technique of in situ hybridization to both meiotic and mitotic chromosomes of Rumex acetosa is described. Differences in the efficiency of signal detection were observed between the two types of material. The implications of these results for in situ hybridization to other plant species are explored.     

Characterization of Charr Chromosomes Using Fluorescence in Situ Hybridization

The chromosomal locations of several families of tandem repetitive DNA sequences and the 5S rDNA were determined using fluorescence in situ hybridization (FISH) in the five North American charr species: Salvelinus namaycush, S. fontinalis, S. alpinus, S. malma, and S. confluentus. The pattern of hybridization of three centromeric repetitive sequences previously isolated from S. namaycush and S. alpinus was unique in each species. Dual-color FISH experiments showed that in several species many of the centromeres had the EcoRI-DraI family in addition to either the AluI-RsaI type A or type B families. The EcoRI-DraI family which was found only at the centromeres of acrocentric chromosomes in S. namaycush, S. fontinalis and S. malma was also found at centromeres of selected metacentrics in S. alpinus (one pair) and S. confluentus (four pairs) whose chromosomes have undergone additional centric fusions compared to the other species. The locations of 5S rDNA sequences were different in each species except for the two most closely related (S. alpinus and S. malma). Two whole-arm chromosome paint probes, one specific for the short and the other for the long arm of the lake charr sex chromosomes, hybridize to the same chromosome pair in all species. Results with other paint probes suggest that independent centric fusions have occurred in S. alpinus and S. confluentus which is consistent with the phylogenetic tree obtained previously for Salvelinus with cytogenetic and DNA data.     

Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml⁻¹ at 37°C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.     

Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection

A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry.In the past decade, Salmonella spp. have been implicated in multiple food-borne disease outbreaks tied to the consumption of fresh fruits and vegetables (19). In the United States, tomatoes have been the most commonly implicated crop for produce-related salmonellosis, with 12 outbreaks occurring since 1998 (3, 19). Contamination of fresh produce can occur at any point in the farm-to-fork continuum and can result from the use of contaminated irrigation water, runoff from adjacent animal production lots, activities of wild animals in fields, or use of untreated manure as a fertilizer (9, 19). Additional routes may include unsanitary practices by workers in the field or even intentional contamination of crops in the field. Although field environments provide greater opportunities for contamination to occur, contamination of tomatoes with Salmonella also occurs for crops grown in controlled (hydroponic) environments (21). The largest documented fresh-produce-related outbreak of salmonellosis to date in the United States occurred during the summer of 2008. Although tomatoes were initially implicated, the source was difficult to pinpoint, and the outbreak strain was later recovered from jalapeño and serrano peppers grown in Mexico. Methods for detection of Salmonella on fresh produce can play an important role in mitigation of disease from outbreaks such as this by providing decision makers with timely data on the presence of this pathogen in contaminated foods.Adhesive-tape-based sampling methods have been used in clinical, environmental, and food microbiology, beginning in the early 1950s (4, 10, 13, 17), and have recently been combined with an rRNA-targeted whole-cell method for fluorescent labeling of specific microbial cells (fluorescence in situ hybridization [FISH]) for culture-independent analysis of microbial communities present on the surfaces of stone monuments (15). We have extended this approach to the rapid sampling of fresh produce surfaces for detection of Salmonella strains, using tomatoes as a model system. In addition to tomatoes, we found that the method could also be used to sample and detect Salmonella artificially inoculated onto jalapeño pepper, cilantro, and spinach surfaces and that cell-charged tapes could be enriched further on Salmonella-selective agar, or in low-volume (0.5 ml) liquid culture followed by flow cytometric analysis.Tomatoes (red tomatoes on the vine, not waxed or oiled; average weight, 135 g), jalapeño peppers, cilantro, and spinach were obtained from a local grocery store and confirmed to be negative for Salmonella via culture. Square regions (1 cm² each) were drawn on produce surfaces with a fine-tip permanent marker using a sterile paper template. Salmonella strains (overnight cultures of serovars Typhimurium ATCC 14028 and Newport, Salmonella Genetic Stock Centre SARB 36, washed and resuspended in 0.1% peptone water) were spot inoculated within each 1-cm² region. Final cell densities ranged from ∼10⁰ to 10⁷ CFU cm⁻². For tomatoes, inocula were applied to skin at either the top (adjacent to the stem scar) or bottom (adjacent to the blossom scar) of the fruit. For spinach and cilantro leaves, the tops of the leaves (adaxial sides) were used. For some samples, mixtures of individual Salmonella strains and Rhodotorula glutinis ATCC 32765 were also spot inoculated in the same fashion (Fig. ​(Fig.1).1). Microbial inocula were allowed to attach by drying onto the tomato surfaces for ∼3 h at 25°C prior to tape-based sampling. Although preliminary experiments suggested that generic office-grade transparent tape may be suitable in this application, we focused on two commercially available adhesive tapes intended for microbiological use: Fungi-Tape (Scientific Device Laboratory, Des Plaines, IL) and Con-Tact-It sampling tape (Birko Corporation, Denver, CO). Microorganisms were sampled by placing Fungi-Tape or Con-Tact-It tape onto inoculated areas, applying gentle and even pressure to ensure full contact of the sampling tape with the produce surface, and removing the tape-cell complex (Fig. ​(Fig.2A).2A). In some experiments, after lifts of cells from tomato surfaces had been made, tapes were placed onto xylose-lysine-Tergitol 4 agar plates, which were then inverted and incubated for 8 h at 37°C for on-tape formation of microcolonies. Following incubation, adhesive tapes were pressed gently against the agar surface to bind any loosely adherent cells, and the tape-cell complex was removed. Prior to further processing (for fixation, hybridization, and microscopy or on-tape liquid culture), cell-charged tapes were mounted (with generic transparent tape) onto microscope slides, sticky side facing upwards. All inoculation and tape-based sampling experiments were repeated three times, using two Salmonella serovars (Typhimurium and Newport); experiments on recovery efficiency of tape-based tomato sampling using serovar Newport were carried out in duplicate and were repeated three times; cytometry experiments were performed twice.Open in a separate windowFIG. 1.Tape-FISH for detection of Salmonella strains in mixed culture from tomato surfaces. Tomatoes were spiked with a mixture of S. enterica serovar Typhimurium (10⁷ CFU cm⁻²) and R. glutinis (10⁶ CFU cm⁻²) and then sampled with adhesive tape after drying. Tapes were hybridized for 30 min with a combination of probes targeting Salmonella cells (Sal3/Salm-63 cocktail, green label) and eukaryotic cells (EUK 516, red label). These results demonstrate the utility of tape-FISH for simultaneous visualization of the distribution and interactions between multiple phylotypes occurring together on produce surfaces.Open in a separate windowFIG. 2.Tape-based sampling of tomato surfaces and liquid surface miniculture. (A) Microorganisms artificially spiked onto tomato surfaces were sampled using sterile adhesive tape. Tapes were applied with gentle and even pressure, ensuring full contact of the sampling tape with produce surfaces, followed by removal of the tape-cell complex for subsequent processing. (B) Filling of a perfusion chamber prior to enrichment via liquid surface miniculture. The bottom surface of the chamber was comprised of a Salmonella-charged tape, mounted sticky side up. After being filled with 500 μl of nonselective broth (TSB or BPW, as described in the text), chambers were incubated for 5 h, followed by cell harvesting, fixation, hybridization, and analysis via flow cytometry (Fig. ​(Fig.33).Liquid phase enrichment (liquid surface miniculture) was performed by placing a CoverWell perfusion chamber (model PC1R-2.0, nonsterile; Grace Bio-Labs, Bend, OR) on top of a slide-mounted tape and filling the chamber with 500 μl growth medium (Trypticase soy broth [TSB] or buffered peptone water [BPW]), preheated to 37°C (Fig. ​(Fig.2B).2B). The flexible silicone base of this type of chamber allowed formation of a water-tight seal, yielding closed, medium-filled chambers whose bottom surfaces were comprised of Salmonella-charged tapes mounted sticky side up on microscope slides. Perfusion chamber inlet ports were sealed using transparent adhesive tape, and the chambers were incubated at 37°C for 5 h.Prior to FISH, tape-bound cells were fixed for 30 min at 25°C by covering the sample contact area with 500 μl of 10% neutral buffered formalin (Sigma). After fixation, the formalin was discarded, and the tape was washed once in 1× phosphate-buffered saline and then dehydrated in ethanol (a 50%, 80%, and 95% series, exposure for 3 min to 300 μl ethanol at each concentration) prior to hybridization. For fixation of liquid surface cultures, the entire 500-μl volume was transferred into a 1.5-ml microcentrifuge tube, pelleted for 5 min at 2,000 × g, resuspended in 0.5 ml 10% buffered formalin, and fixed for 30 min at 25°C. Fixed samples were harvested via centrifugation (5 min, 2,000 × g), the supernatant was discarded, and cell pellets were resuspended in 0.5 ml of cell storage solution (a 50:50 mix of phosphate-buffered saline-absolute ethanol) and either analyzed directly or stored at −20°C until analyzed.Two oligonucleotide probes previously developed for detection of Salmonella spp., Sal3 (20) and Salm-63 (14), were combined as described by Lantz et al. (18) and applied as a dual probe cocktail at a total concentration of 5 ng/μl probe (2.5 ng/μl each probe). In mixed-flora experiments with R. glutinis, a universal Eucarya probe, EUK 516 (1), was also used at 5 ng/μl. Probes were synthesized and high-pressure liquid chromatography purified by Integrated DNA Technologies (Coralville, IA) and were labeled at the 5′ end with fluorescein or Texas Red (for microscopy work) or with Cy5 (for flow cytometry experiments). For most experiments, samples on tapes were hybridized for 15 min at 55°C using a moisture-sealed slide incubation chamber (Slide Moat model 240000; Boekel Scientific, Feasterville, PA). Briefly, 300-μl volumes of hybridization buffer (0.7 M NaCl, 0.1 M Tris [pH 8.0], 0.1% sodium dodecyl sulfate, 10 mM EDTA, containing probe and preheated to 55°C) were applied to the surface of the tape, and the chamber''s lid was sealed, creating a moist, temperature-controlled environment within the chamber. After 15 min, the lid was removed, and samples were briefly rinsed with probe-free hybridization buffer, which had been preheated to 55°C. Tapes were then processed for microscopy, as described below. In initial tests, and for Fig. ​Fig.1,1, hybridization and washing (30 min each) were carried out in a hybridization oven (Bambino; Boekel Scientific), inside sealed 50-ml polypropylene centrifuge tubes. Due to the limited throughput of this approach, subsequent hybridizations were carried out using the Slide Moat, which allowed analysis of multiple (>20) slides and also provided direct-contact heat transfer. For hybridization of cells grown using liquid surface miniculture, fixed cells (entire 500-μl samples, in cell storage solution) were pelleted (5 min, 2,000 × g) and resuspended in 100 μl of probe-containing hybridization buffer. Samples were hybridized at 55°C on a heat block for 30 min, followed by a 30-min wash step at the same temperature using 500 μl hybridization buffer without probe, and then analyzed via cytometry.Hybridized cells on tapes were counterstained for 10 min in the dark with ∼30 μl mounting medium containing 1.5 μg ml⁻¹ DAPI (Vectashield H-1200; Vector Laboratories, Burlingame, CA) and then mounted with a coverslip and examined using a Leitz LaborLux S microscope equipped with a Canon PowerShot A640 consumer-grade digital camera controlled by Axiovision software (v. 4.6; Carl Zeiss Microimaging, Inc., Thornwood, NY). Raw TIFF outputs from green (fluorescein) and red (Texas Red) channels were adjusted for brightness and contrast to appear as they did via microscopy, and composite images were made using Adobe Photoshop. Flow cytometry of liquid surface miniculture samples was performed on a Becton-Dickinson FACSCanto flow cytometer with red (647-nm) excitation, using bacterial side scatter to trigger event detection. Samples were run for 3 min at a low flow rate (10 μl min⁻¹). Flow cytometry data were analyzed using FlowJo software (v. 8.7.1; Tree Star Inc., Ashland, OR).Since its introduction in 1930, “Scotch”-type adhesive tape has been adopted for a number of “off-label” uses, including use in the household for removal of lint from garments, in forensic science for lifting fingerprints from surfaces, and in the clinic for sampling and detection of intestinal parasites or their eggs via anal tape lifts or for sampling of pathogenic fungi from skin (4, 10). In environmental microbiology, adhesive tape has been used for sampling of microbes from leaf surfaces for subsequent microscopic or cultural analyses (17), and tape-based sampling is an accepted technique in food microbiology for monitoring of food or environmental surfaces (12, 13). For example, the use of Con-Tact-It tape is suggested in the Compendium of Methods for the Microbiological Examination of Foods (12) as an alternative to RODAC plating for estimating the sanitary condition of food processing environmental surfaces (12), and use of this tape has also been combined with acridine orange staining for sampling and analysis of microbial populations on beverage dispenser tips via fluorescence microscopy (16). Extending the approach further, La Cono and Urzì (15) combined tape-based sampling with on-tape FISH for the detection and characterization of microflora present on the surfaces of historic stone monuments and suggested the approach for use on other surfaces, including food contact surfaces. However, in addition to inanimate objects (i.e., cutting boards, countertops, floor tiles, processing equipment, etc.), the surfaces of many foods themselves may become contaminated with human pathogens. In the United States, tomatoes and other fresh produce have been implicated in a number of recent outbreaks of salmonellosis, therefore, we sought to examine the utility of this tape-FISH approach for sampling and direct detection of Salmonella strains on tomato and other fresh produce surfaces.We found that two commercially available microbiological sampling tapes (Fungi-Tape and Con-Tact-It) could be used to remove Salmonella strains and other microorganisms from the surfaces of tomatoes (with greater than 99% recovery efficiency determined for S. enterica serovar Newport at an inoculum level of 10⁷ CFU cm⁻² using Fungi-Tape [data not shown]) and that Salmonella cells could be detected via FISH performed directly on the tape. Use of this tape-FISH approach was also demonstrated for other types of produce considered at risk for contamination with Salmonella spp., including jalapeño peppers, spinach, and cilantro (data not shown). The limit of direct detection via fluorescence microscopy was 10³ CFU cm⁻²—the practical limit of detection for manual microscopy (2)—and all procedures (surface sampling, cell fixation, dehydration, hybridization, counterstaining, and detection) could be carried out within ∼1.5 h. We also found that salmonellae could be enriched at a tape-agar interface by simply laying cell-charged tapes face down on selective agar plates. Substantial microcolony formation was observed after only 8 h at 37°C (data not shown). Alternatively, nonsterile perfusion chambers could be sealed over slide-mounted sampling tapes, allowing liquid surface miniculture-based enrichment of sampled cells in nonselective broths. TSB was superior to BPW, both in its ability to support the growth of Salmonella strains and in promoting release from the tape into liquid miniculture (Fig. ​(Fig.3).3). Although the ultimate level of detection was not determined for the combination of liquid surface microculture and flow cytometry, a relatively small number of cells (10³ cm⁻²) could be detected directly from TSB-washed tapes, and substantial enrichment of Salmonella strains was observed after a brief enrichment in liquid surface miniculture (500-μl volumes, 5 h of enrichment at 37°C), even in the absence of visible turbidity (Fig. ​(Fig.3).3). Our work highlights the potential for tape-FISH to provide rapid and specific detection of Salmonella spp. on fresh produce surfaces, even in the presence of nontarget organisms.Open in a separate windowFIG. 3.Tape-FISH combined with liquid surface miniculture for rapid detection of S. enterica serovar Typhimurium on tomatoes via flow cytometry. Adhesive tape was used to remove serovar Typhimurium from tomato surfaces inoculated with 10³ cells cm⁻². As described for Fig. ​Fig.2B,2B, cell-charged tapes were mounted face-up on microscope slides, and perfusion chambers were placed on top of the tape and filled with nonselective broths. Liquid surface minicultures were incubated for 5 h, mixed via up-and-down pipetting using gel loading tips, processed for FISH, and then analyzed via flow cytometry. When TSB was used, it was possible to detect Salmonella directly from tapes (0 h, TSB). Despite a lack of visible turbidity, substantial enrichment was possible after only 5 h of nonselective pre-enrichment in TSB. These data show the utility of FISH and flow cytometry in combination with adhesive-tape-based sampling for the rapid detection of Salmonella on contaminated tomatoes.As a simple approach for sampling, adhesive-tape methods have a number of potential advantages: they are easy to learn, use, and troubleshoot, and the raw materials or equipment needed are inexpensive and widely available. They are portable enough to facilitate testing in the field or in a food production environment and are nondestructive (15). Because they have the potential to save both time and money, use of such simple methods may free up limited resources, enabling more frequent or extensive testing. Additionally, tape-based sampling comprises elements of both sample preparation and sample presentation. That is, the same action (contact with the food surface) accomplishes both removal of attached organisms from the surface and two-dimensional presentation of the cells on an optically clear film, facilitating downstream processing, such as staining (colorimetric staining, fluorescent staining, and FISH) and direct examination via microscopy. Of special benefit to FISH-based analyses is the fact that microbial cells are removed from the host tissues, which could be a significant source of interference with probe-conferred fluorescence, due to the intense autofluorescence often seen in plant tissues (5, 6, 8).Tape-based detection approaches have long been used in environmental microbiology for examination of plant-associated microorganisms, such as fungi present on leaves (10, 17). A key benefit of this application is that the spatial relationships of the sampled organisms from the leaves are preserved as a “mirror image” in situ on the tape (15, 17). The FISH approach has been used to great advantage in environmental microbiology for cultivation-independent analyses of complex microbial consortia, and FISH has also been a valuable tool for studying the spatial arrangements and physical interactions of specific microbes occurring in foods, such as artisanal cheeses (7, 11). Because FISH is a culture-independent approach, tape-FISH can theoretically be used for in situ examination of target cells on fruit or leaf surfaces, without the need for culture. Because multiple probes can be used, the presence and physical location of more than one phylotype can be determined and monitored simultaneously (Fig. ​(Fig.11).In their study on the colonization of cilantro leaves by S. enterica serovar Thompson, Brandl and Mandrell (5) found that low inocula of this organism were able to reach high cell densities when the leaves were stored under humid conditions. S. enterica serovar Thompson formed distinct microcolonies or large mixed-species aggregates with other enteric species commonly found as epiphytes on cilantro, such as Pantoea agglomerans. In the study of Barak and Liang (3), cocolonization of tomato plants with the plant pathogen Xanthomonas campestris pv. vesicatoria led to significantly higher populations of S. enterica than on plants colonized by S. enterica alone, suggesting cooperative activities of these two organisms during growth on these plants. Metabiotic interactions between proteolytic molds and Salmonella spp. have also been documented for raw, ripe tomatoes, with the metabolic activities of spoilage molds and concomitant physical degradation of tomato surfaces enhancing the growth of S. enterica (23). In light of these studies, culture-independent techniques capable of preserving spatial information on relationships between target cells, competitive or cooperative microflora, and host structures are expected to be of great value to basic research on pathogen-produce interactions. Our tape-FISH protocol may therefore be leveraged as a basic research tool and, when coupled with enrichment, as a rapid and simple approach for sampling and screening for Salmonella on fruit, herb, or leafy greens surfaces in support of routine control measures or as a tool for outbreak investigation.Several factors can potentially impact the efficiency of cell capture or release by the tape, including serovar-dependent differences in cell surface properties, the mode of attachment (i.e., nonspecific adhesion or adhesion mediated by specific structures, such as pili or flagella), the presence of soil on or moisture content of the sample surface, and whether microbial cells are present in a monolayer or in a firmly attached biofilm (6, 13, 17, 22). Because different brands of commercially available tapes are expected to be formulated with different adhesives, they may also vary in their adhesive properties or compatibility with living cells, which could also impact cell recovery, release, or growth. As noted, we were able to recover serovar Newport artificially inoculated at 10⁷ CFU ml⁻¹ onto untreated tomatoes (no waxes or oils) with greater than 99% efficiency using Fungi-Tape, and cells remained culturable, as determined by agar and liquid surface miniculture enrichment.As a sampling method, tape-based removal of microorganisms from vegetable surfaces faces some practical challenges. In principle, FISH is capable of single-cell sensitivity, but, as noted in a review by Amann et al. (2), bringing a single FISH-labeled cell into view under the microscope is technically challenging, with an inverse relationship existing between the number of target cells present and the time needed to find them. Therefore, rapid and reliable detection of fewer than 10³ cells per cm² is not practical using manual microscopy (2), a result that we confirmed for tape-FISH in our work. This is expected to remain a limitation of simple, manual microscopy, but developments in automated microscopy or use of scanning laser cytometry could be effective means for reliable identification of lower levels of target cells occurring on hybridized tapes.One potential limitation of our tape-FISH approach is that salmonellae may be randomly distributed over produce surfaces and might be missed, depending on which surface is tested. In the testing of beef carcasses, sampling is narrowed to well-defined regions (i.e., brisket, flank, rump) previously established to harbor the highest microbial loads. In testing of certain types of produce, it may therefore be possible to focus sampling on well-defined regions of plant surfaces that may preferentially harbor Salmonella spp., such as the vein structures on cilantro leaves or the stem scar of tomatoes (5). The use of such rational sampling approaches may increase the likelihood of detecting Salmonella spp. or other pathogens on the surfaces of some types of produce via tape-FISH.Tape-based sampling methods have long been used in the separate fields of environmental, food, and clinical microbiology. Therefore it is fitting to recognize that tape-FISH, as described here, may have potential applications at various points along the production-to-consumption-to-disease (or farm-to-fork-to-physician) continuum. We have described the use of tape-FISH for detection of Salmonella strains on the surfaces of tomatoes, jalapeños, spinach, and cilantro and have shown for tomatoes that this dual sampling and sample presentation approach can also be combined with brief enrichments using either Salmonella-selective agar (xylose-lysine-Tergitol 4) or nonselective-broth (TSB) culture. In the latter application, we found that because the tape-cell complex is essentially two dimensional, we could perform a liquid surface miniculture step by overlaying a minimal volume of broth on the tape after it was affixed to a microscope slide. In this application, tape-based sampling effectively represents a means for cell concentration prior to enrichment. Enrichment of even relatively few cells in a small volume with subsequent analysis of the entire volume may be a promising means for facilitating earlier detection of target cells, as no subsequent concentration step (filtration, centrifugation, etc.) is needed. In addition to its use for detection, the tape-FISH technique may also be a valuable research tool for exploring events occurring during the colonization of tomatoes by Salmonella, or the interplay between spoilage microflora and Salmonella and the role of such metabiotic interactions on establishment and persistence of infection (3, 23). It is hoped that the established and familiar nature of adhesive-tape-based techniques combined with our simple and streamlined approach for FISH-based staining of target cells will enable more rapid adoption of the tape-FISH approach by food microbiologists who may not be familiar with or currently using whole-cell molecular techniques.     

荧光原位杂交技术在慢性淋巴细胞白血病遗传学异常检测中的应用

目的:分析在荧光原位杂交技术慢性淋巴细胞白血病遗传学异常检测中的应用,并分析相关指标在评价患者预后中的应用。方法:对我院收治的45例初诊CLL患者采用荧光原位杂交技术进行特异性探针D13S25(13q14.3)、RB1(13q14)、p53(17p13)、ATM(11q22.3)、以及CSP12(12号染色体3体)染色体标本检测,分析CLL患者遗传学异常的发生率。采用实时定量PCR检测miR-15a和miR-16-1与CLL患者遗传学异常的相关性。结果:45例CLL初诊患者中,荧光原位检测发现CLL遗传学异常37例,CLL遗传学异常率82.22%。其中d(13q14.3)遗传异常13例,d(13q14)遗传异常7例,d(11q22-23)遗传异常6例,d(17p13)遗传异常5例,12号染色体三体异常6例,遗传学异常多呈异质性。实时定量PCR检测发现miR-15a和miR-16-1与d(13q14)遗传异常显著相关。结论:荧光原位杂交技术是一种检测CLL遗传学异常的快速、灵敏方法,可以提高CLL遗传异常检出率。miR-15a和miR-16-1可以预测d(13q14)遗传异常CLL患者预后。     

Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting

Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.     

Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization

Background

There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders ( = Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination.

Methodology/Principal Findings

To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4′, 6-diamidino-2-phenylindole (DAPI). The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes.

Conclusions/Significance

Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future.     

Hybrid Identification in Clivia(Amaryllidaceae) using Chromosome Banding and Genomic In Situ Hybridization

Giemsa C-banding and genomic in situ hybridization (GISH) wereused to identify parental genomes in hybrids of Clivia(Amaryllidaceae).Of the three groups reputed to be hybrids, onlyC. cyrtanthiflorawas shown to be of hybrid origin. The ‘German hybrids’and ‘Belgian hybrids’ were both shown to be karyotypicallyand genomically similar to C. miniata, and are either selectionsor intraspecific hybrids of that species. Successful genomedifferentiation in F₁hybrids by GISH required high stringencyand high ratios of blocking DNA to probe. The spatial dispositionof different genomes with C-band or GISH markers in the hybridswas investigated in two dimensions on the spread. In five artificiallyproduced hybrids, either C-banding or GISH was used to locatethe position of parental genomes in mitotic metaphase cells.In all cases there was a significant tendency for centromeresof the different parental genomes to occupy two distinct concentricdomains on the metaphase plate. The presence or absence of centromericheterochromatin was not correlated with genome disposition.Results show that chromosome analyses can be a useful way ofidentifying Clivia hybrids in their vegetative phase. Copyright2001 Annals of Botany Company Clivia, genomic in situ hybridization, cultivar origin, parental genome separation     

Development of a Two-Color Fluorescence In Situ Hybridization Technique for Species-Level Identification of Human-Infectious Cryptosporidium spp.

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.Cryptosporidium (Apicomplexa) is a genus of protozoan parasites with species and genotypes that infect humans, domesticated livestock, companion animals, and wildlife worldwide (5, 6, 14, 15, 20, 23). The majority of cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or C. hominis (8, 10, 19, 24), although rare cases due to species such as C. meleagridis, C. felis, or C. canis have been reported (8, 9, 11-13, 17, 18, 22). The specific identification and characterization of Cryptosporidium species are central to the control of this disease in humans and a wide range of animals.One of the most widely adopted techniques for the identification of microorganisms in complex microbial communities is fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes (2-4). This method relies on the hybridization of synthetic oligonucleotide probes to specific regions within the rRNA of the organism. While FISH has been applied for the detection of Cryptosporidium oocysts in water samples (21), no FISH probes that successfully differentiate C. hominis from C. parvum have been reported.We have reported previously on the design of a species-specific probe, Cpar677, that detects C. parvum (1). In this study, we report on the design and validation of a C. hominis species-specific probe, Chom253. Together, the two probes were used here for the development of a two-color, microscopy-based FISH assay for the simultaneous detection of C. parvum and C. hominis.     

原位杂交技术及其在果树研究中的应用

原位杂交技术是近年来快速发展起来的一门新技术,本文介绍了原位杂交技术的基本原理、方法及其发展前景,以及该技术与其它生物学技术相结合而形成的一些新技术。综述了这些技术在果树研究中的应用情况。     

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.     

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Characterization of Eastern Oyster (Crassostrea virginica Gmelin) Chromosomes by Fluorescence In Situ Hybridization with Bacteriophage P1 Clones

Chromosome identification is an essential step in genomic research, which so far has not been possible in oysters. We tested bacteriophage P1 clones for chromosomal identification in the eastern oyster Crassostrea virginica, using fluorescence in situ hybridization (FISH). P1 clones were labeled with digoxigenin-11-dUTP using nick translation. Hybridization was detected with fluorescein-isothiocyanate-labeled anti-digoxigenin antibodies and amplified with 2 layers of antibodies. Nine of the 21 P1 clones tested produced clear and consistent FISH signals when Cot-1 DNA was used as a blocking agent against repetitive sequences. Karyotypic analysis and cohybridization positively assigned the 9 P1 clones to 7 chromosomes. The remaining 3 chromosomes can be separated by size and arm ratio. Five of the 9 P1 clones were sequenced at both ends, providing sequence-tagged sites that can be used to integrate linkage and cytogenetic maps. One sequence is part of the bone morphogenetic protein type 1b receptor, a member of the transforming growth factor superfamily, and mapped to the telomeric region of the long arm of chromosome 2. This study shows that large-insert clones such as P1 are useful as chromosome-specific FISH probes and for gene mapping in oysters.     

体外诱导分化神经细胞的原位分子杂交方法

经１×１０－６ｍｏｌ／Ｌ视黄酸诱导的Ｐ１９细胞体外可向神经方向分化,接种于多聚赖氨酸（ｐｏｌｙＤｌｙｓｉｎｅ）和纤连蛋白（ｆｉｂｒｏｎｅｃｔｉｎ）包被的玻片后,细胞逐渐聚集成团,此时细胞的贴壁性较差,进行原位分子杂交时容易脱落。我们尝试在细胞表面覆盖一层明胶,减少了细胞的脱落,又比较了蛋白酶Ｋ和胃蛋白酶对细胞蛋白质的消化作用,确定胃蛋白酶可较温和地消化细胞蛋白质,使探针有效地透入结合,杂交后细胞亦能较完整地保留于玻片上。     

Mechanistic Approach to the Problem of Hybridization Efficiency in Fluorescent In Situ Hybridization

In fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting. Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only. In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed. The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (ΔG°_overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH. Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA of Escherichia coli were used to demonstrate that ΔG°_overall is a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex. The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA. Finally, a threshold ΔG°_overall of −13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.     

RNA—RNA原位杂交实验条件探讨

将Vigilin和qroal(Ⅰ)cDNA亚克隆到pGEM3Z和pGEM4Z载体,体外转录合成35s标记的cRNA探针。经RNA凝胶电泳,Southern Northern,杂交检查探针长度,杂交特性和特异性,通过系列实验探讨了RNA-RNA原位杂交实验中固定、杂交前处理、杂交温度,探针量、探针长度,洗脱严格性和RNA酶处理等对杂交结果的影响,建立了简化的RNA-RNA原位杂交方法。