Time-resolved fluorescence detection of enzyme-amplified lanthanide luminescence for nucleic acid hybridization assays

1991 ◽  
Vol 37 (9) ◽  
pp. 1506-1512 ◽  
Author(s):  
E F Templeton ◽  
H E Wong ◽  
R A Evangelista ◽  
T Granger ◽  
A Pollak
Keyword(s):  
Alkaline Phosphatase ◽  
Nucleic Acid ◽  
Fluorescence Detection ◽  
Dna Hybridization ◽  
Nucleic Acid Hybridization ◽  
Time Resolved Fluorescence ◽  
Dot Blot ◽  
Time Resolved ◽  
Lanthanide Luminescence ◽  
Hybridization Assays

Abstract A new nonisotopic detection method based on time-resolved fluorescence for nucleic acid hybridization assays with alkaline phosphatase labels has been developed: enzyme-amplified lanthanide luminescence (EALL). EALL combines the amplification of an enzyme label with the sensitivity and background elimination of time-resolved fluorescence detection of lanthanide ion luminescence. The detection system for alkaline phosphatase makes use of a phosphorylated salicylic acid derivative that, upon dephosphorylation, gives a product capable of forming a luminescent terbium chelate. We demonstrate DNA hybridization assays by using two substrates, one for membrane and one for solution-based formats. Using the substrate that produces a more adhesive product allows performance of dot-blot and Southern blot assays on nylon membranes; results can be recorded with a time-resolved photographic camera system, or with an ultraviolet transilluminator-based system. Less than 4 pg of target sequence can be detected in a dot-blot assay after incubation with substrate for 2-4 h. DNA microwell-plate hybridization assays with the more soluble substrate/product pair can be quantified with time-resolved fluorescence plate readers, giving a similar detection sensitivity. EALL is thus a practical time-resolved fluorescence-based alternative to other detection systems for DNA hybridization assays.

scholarly journals Characterization of a Severe Strain of Cucumber Mosaic Cucumovirus Seedborne in Cowpea

Plant Disease ◽  
1998 ◽  
Vol 82 (4) ◽  
pp. 419-422 ◽  
Author(s):  
A. G. Gillaspie ◽  
M. R. Hajimorad ◽  
S. A. Ghabrial
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Hybridization ◽  
Satellite Rna ◽  
Severe Strain ◽  
Cucumber Mosaic Cucumovirus ◽  
Hybridization Assays

A new seedborne strain of cucumber mosaic cucumovirus (CMV) that induces severe symptoms on many cowpea genotypes was detected in Georgia in 1994. This strain, designated CMV-Csb, is asymptomatic on tobacco, but it produces more severe cowpea stunt symptoms when present in combination with blackeye cowpea mosaic potyvirus than do the more prevalent CMV isolates. The new strain is seedborne in cowpea (1.5 to 37%), has no associated satellite RNA, and is classified as a member of subgroup I of CMV strains based on nucleic acid hybridization assays.

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

1990 ◽  
Vol 36 (9) ◽  
pp. 664-669 ◽  
Author(s):  
David R. Preston ◽  
G. Rasul Chaudhry ◽  
Samuel R. Farrah
Keyword(s):  
Tissue Culture ◽  
Nucleic Acid ◽  
Environmental Samples ◽  
Nucleic Acid Hybridization ◽  
Dot Blot ◽  
Polio Virus ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Hybridization Procedure ◽  
Great Specificity

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

scholarly journals Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes

1988 ◽  
Vol 251 (3) ◽  
pp. 935-938 ◽  
Author(s):  
A H al-Hakim ◽  
R Hull
Keyword(s):  
Nucleic Acid ◽  
Blot Hybridization ◽  
Nucleic Acid Hybridization ◽  
Cross Linking ◽  
Dot Blot ◽  
Hybridization Probes ◽  
Target Dna ◽  
Amino Derivatives ◽  
Biotin Molecule ◽  
Long Chain

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.

Detection of Escherichia coli rRNA using target amplification and time-resolved fluorescence detection

1991 ◽  
Vol 5 (6) ◽  
pp. 467-472 ◽  
Author(s):  
Charlene E. Bush ◽  
Kurt M. Vanden Brink ◽  
David G. Sherman ◽  
W. Richard Peterson ◽  
Laura A. Beninsig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Detection ◽  
Time Resolved Fluorescence ◽  
Time Resolved
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