scholarly journals Oligonucleotides as Hybridization Probes to Localize Phytoplasmas in Host Plants and Insect Vectors

1999 ◽  
Vol 89 (10) ◽  
pp. 894-901 ◽  
Author(s):  
D. R. Webb ◽  
R. G. Bonfiglioli ◽  
L. Carraro ◽  
R. Osler ◽  
R. H. Symons
Keyword(s):  
Plant Tissue ◽  
Multiple Infections ◽  
Oligonucleotide Probes ◽  
Vector Ecology ◽  
Tetrazolium Chloride ◽  
Detection Systems ◽  
Hybridization Probes ◽  
Euscelidius Variegatus ◽  
Polymerase Chain Reaction Primers ◽  
Clover Phyllody

Protocols have been developed using 20- to 24-mer oligodeoxynucleotides, originally designed as polymerase chain reaction primers, as hybridization probes for the nonradioactive detection of Italian clover phyllody (ICPh) phytoplasma in plant (Chrysanthemum carinatum) and leafhopper (Euscelidius variegatus) tissue. In situ hybridization of paraffin-embedded tissue sections was carried out using oligodeoxynucleotides 5′ end-labeled with either Cy5 fluorochrome, biotin, or digoxigenin. The Cy5-labeled oligonucleotide probes that hybridized to phytoplasmas present in plant tissue were visualized by confocal microscopy. The biotin- and digoxigeninlabeled probes were detected in both plant and insect tissue using a chromogenic alkaline phosphatase-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate reaction. An enhancement of a signal was observed in plant tissue when a tyramide signal-amplification procedure was incorporated into the biotin or digoxigenin detection systems. The results obtained using these techniques with the ICPh phytoplasma system showed that they can provide a rapid means of confirming vector status in insects. Due to the potential ability of short, labeled, oligonucleotide probes to specifically distinguish between different phytoplasmas present in multiple infections, this technique should provide a powerful new tool for epidemiological and vector ecology studies.

Characterization of filamentous foaming in activated sludge systems using oligonucleotide hybridization probes and antibody probes

1998 ◽  
Vol 37 (4-5) ◽  
pp. 485-493 ◽  
Author(s):  
Francis L. de los Reyes ◽  
Daniel B. Oerther ◽  
Ma. Fiorella de los Reyes ◽  
Mark Hernandez ◽  
Lutgarde Raskin
Keyword(s):  
Activated Sludge ◽  
Oligonucleotide Probes ◽  
Mixed Liquor ◽  
Antibody Staining ◽  
Hybridization Probes ◽  
Oligonucleotide Hybridization ◽  
Pure Cultures ◽  
In Situ Hybridizations ◽  
Filamentous Foaming

A quantitative method was developed for estimating Gordona mass in activated sludge foam and mixed liquor samples. The technique involves in situ hybridization with a genus-specific fluorescently labeled oligonucleotide probe calibrated on pure cultures of Gordona. The immunofluorescent technique of Hernandez et al. was modified to allow staining with fluorescently labeled antibody and hybridization probes. The results of this technique were compared to those from membrane hybridization studies using radioactively-labeled oligonucleotide probes. Quantitative membrane hybridizations, in situ hybridizations, and antibody staining resulted in significantly different levels of Gordona in activated sludge foam, activated sludge mixed liquor, return activated sludge, and anaerobic digester sludge. Simultaneous staining with labeled antibodies and oligonucleotide probes provide a definitive identification for Gordona, and represents a new approach for in situ studies of this organism's role in foaming.

Enzyme-labeled oligonucleotide probes for detection of the genes for theromostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus

1992 ◽  
Vol 38 (5) ◽  
pp. 410-416 ◽  
Author(s):  
Koichiro Yamamoto ◽  
Takeshi Honda ◽  
Toshio Miwatani ◽  
Shigeru Tamatsukuri ◽  
Shuji Shibata
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Recombinant Dna ◽  
Bacterial Species ◽  
Blot Hybridization ◽  
Oligonucleotide Probes ◽  
Dot Blot ◽  
Hybridization Probes ◽  
Specificity And Sensitivity ◽  
Thermostable Direct Hemolysin ◽  
Dna Fragment

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh. Key words: oligonucleotide DNA, enzyme, Vibrio parahaemolyticus, hemolysin.

Freeze-fracture, freeze-etch complementary pairs of virus-infected cells reveal value of etching even when relatively high concentrations of cryoprotectants are used

1978 ◽  
Vol 36 (2) ◽  
pp. 136-137
Author(s):  
Russell L. Steere ◽  
Eric F. Erbe
Keyword(s):  
Plant Tissue ◽  
Phosphate Buffer ◽  
Infected Plant ◽  
Freeze Fracture ◽  
Distilled Water ◽  
Virus Infected Plant ◽  
Infected Cells ◽  
High Concentrations

It has been assumed by many involved in freeze-etch or freeze-fracture studies that it would be useless to etch specimens which were cryoprotected by more than 15% glycerol. We presumed that the amount of cryoprotective material exposed at the surface would serve as a contaminating layer and prevent the visualization of fine details. Recent unexpected freeze-etch results indicated that it would be useful to compare complementary replicas in which one-half of the frozen-fractured specimen would be shadowed and replicated immediately after fracturing whereas the complement would be etched at -98°C for 1 to 10 minutes before being shadowed and replicated.Standard complementary replica holders (Steere, 1973) with hinges removed were used for this study. Specimens consisting of unfixed virus-infected plant tissue infiltrated with 0.05 M phosphate buffer or distilled water were used without cryoprotectant. Some were permitted to settle through gradients to the desired concentrations of different cryoprotectants.

The Potentials of Scanning Microscopy

1968 ◽  
Vol 26 ◽  
pp. 352-353
Author(s):  
A. V. Crewe
Keyword(s):  
Salient Feature ◽  
Secondary Emission ◽  
Resolving Power ◽  
X Rays ◽  
Scanning Microscope ◽  
Forming Process ◽  
Additional Tool ◽  
Detection Systems ◽  
Conventional Microscope ◽  
Present Information

If the resolving power of a scanning electron microscope can be improved until it is comparable to that of a conventional microscope, it would serve as a valuable additional tool in many investigations.The salient feature of scanning microscopes is that the image-forming process takes place before the electrons strike the specimen. This means that several different detection systems can be employed in order to present information about the specimen. In our own particular work we have concentrated on the use of energy loss information in the beam which is transmitted through the specimen, but there are also numerous other possibilities (such as secondary emission, generation of X-rays, and cathode luminescence).Another difference between the pictures one would obtain from the scanning microscope and those obtained from a conventional microscope is that the diffraction phenomena are totally different. The only diffraction phenomena which would be seen in the scanning microscope are those which exist in the beam itself, and not those produced by the specimen.

Universal ESEM

1993 ◽  
Vol 51 ◽  
pp. 786-787
Author(s):  
G.D. Danilatos
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
High Vacuum ◽  
Natural Extension ◽  
Necessary Condition ◽  
Environmental Scanning ◽  
Detection Systems ◽  
Small Gain ◽  
Detection Medium ◽  
Scanning Electron

The environmental scanning electron microscope (ESEM) has evolved as the natural extension of the scanning electron microscope (SEM), both historically and technologically. ESEM allows the introduction of a gaseous environment in the specimen chamber, whereas SEM operates in vacuum. One of the detection systems in ESEM, namely, the gaseous detection device (GDD) is based on the presence of gas as a detection medium. This might be interpreted as a necessary condition for the ESEM to remain operational and, hence, one might have to change instruments for operation at low or high vacuum. Initially, we may maintain the presence of a conventional secondary electron (E-T) detector in a "stand-by" position to switch on when the vacuum becomes satisfactory for its operation. However, the "rough" or "low vacuum" range of pressure may still be considered as inaccessible by both the GDD and the E-T detector, because the former has presumably very small gain and the latter still breaks down.

Freeze-Fracture Studies of Membranes in Developing Sieve Elements in a Plant Tissue Culture

1980 ◽  
Vol 38 ◽  
pp. 636-637
Author(s):  
R. D. Sjolund ◽  
C. Y. Shih
Keyword(s):  
Plant Tissue ◽  
Cultured Cells ◽  
Freeze Fracture ◽  
Tissue Cultures ◽  
Sieve Elements ◽  
Intact Plants ◽  
Plant Tissue Cultures ◽  
Whole Plants ◽  
Energy Dependent ◽  
Similar Elements

The differentiation of phloem in plant tissue cultures offers a unique opportunity to study the development and structure of sieve elements in a manner that avoids the injury responses associated with the processing of similar elements in intact plants. Short segments of sieve elements formed in tissue cultures can be fixed intact while the longer strands occuring in whole plants must be cut into shorter lengths before processing. While iyuch controversy surrounds the question of phloem function in tissue cultures , sieve elements formed in these cultured cells are structurally similar to those of Intact plants. We are particullarly Interested In the structure of the plasma membrane and the peripheral ER in these cells because of their possible role in the energy-dependent active transport of sucrose into the sieve elements.

Sign in / Sign up

Export Citation Format

Share Document