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.

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.

Comparative Studies of Nucleic Acid Hybridization Assay for Listeria in Foods

1988 ◽  
Vol 71 (3) ◽  
pp. 669-673 ◽  
Author(s):  
Jeffrey D Klinger ◽  
Andrew Johnson ◽  
Daniel Croan ◽  
Pauline Flynn ◽  
Kevan Whippie ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Environmental Samples ◽  
False Negative ◽  
False Negative Rate ◽  
Nucleic Acid Hybridization ◽  
Hybridization Assay ◽  
Rapid Screening ◽  
Bacterial Strains ◽  
Synthetic Dna ◽  
Conventional Culture

Abstract A nucleic acid hybridization assay has been developed for Listeria spp. in dairy foods and environmental samples. The assay is based on detection of unique Listeria 16S rRNA sequences by using a 32Plabeled synthetic DNA probe. Inclusivity and exclusivity of the probe were confirmed with 139 Listeria isolates representing all known species, and 73 non-Listeria bacterial strains. In this paper, we present results from our preliminary studies comparing the hybridization assay with conventional culture on a total of 575 specimens that represent a variety of inoculated and uninoculated foods and environmental samples. The assay, which is done in a filter manifold format after 2 days of cultural enrichment, requires a total assay time of less than 2.5 days. The false-negative rate for all sample groups tested using the GENE-TRAK hybridization assay was less than the rate for culture. Thus, the new assay allows rapid screening of the indicated product groups and provides reliable numerical results

A SURVEY OF THE INCIDENCE OF POTATO SPINDLE TUBER VIROID IN PRINCE EDWARD ISLAND USING TWO TESTING METHODS

1988 ◽  
Vol 68 (4) ◽  
pp. 1229-1236 ◽  
Author(s):  
R. P. SINGH ◽  
T-L. DeHAAN ◽  
A. S. JASWAL
Keyword(s):  
Nucleic Acid ◽  
Gel Electrophoresis ◽  
Prince Edward Island ◽  
Potato Crop ◽  
Polyacrylamide Gel Electrophoresis ◽  
Leaf Disc ◽  
Potato Spindle Tuber Viroid ◽  
Polyacrylamide Gel ◽  
Nucleic Acid Hybridization ◽  
Dot Blot

Return-polyacrylamide gel electrophoresis (R-PAGE) and nucleic acid hybridization (dot-blot) were used for the detection of potato spindle tuber viroid (PSTV) from potato leaves. Both methods detected potato plants experimentally infected in the first season or those produced from infected tubers (secondarily infected). PSTV concentration was lower in the first-season infected plants than those in the second. Both methods detected PSTV in a single leaf disc from field-grown infected plants combined with 399 – 499 discs from field-grown healthy plants. The sensitivity of detection by R-PAGE was lower for certain cultivars and increased with the age of plants. About 85 000 leaf samples collected from 123 tablestock fields, 170 seed fields, and 63 cultivars from the Fox Island Elite Seed Farm in Prince Edward Island were found to be free from PSTV infection. Reasons for PSTV absence in the potato crop are discussed.Key words: Diagnosis, dot-blot, return polyacrylamide gel electrophoresis, nucleic acid hybridization, Solanum tuberosum, potato

Comparison of Poliovirus Detection in Water by Cell Culture and Nucleic Acid Hybridization

1993 ◽  
Vol 27 (3-4) ◽  
pp. 315-319
Author(s):  
Carlos E. Enriquez ◽  
Morteza Abbaszadegan ◽  
Ian L. Pepper ◽  
Kenneth J. Richardson ◽  
Aaron B. Margolin ◽  
...  
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Nucleic Acid ◽  
Water Samples ◽  
Environmental Water ◽  
Nucleic Acid Hybridization ◽  
Well Water ◽  
Gene Probe ◽  
Hybridization Technique ◽  
Water Filter

The nucleic acid hybridization technique has been used to detect viral nucleic acid in environmental water samples. This type of assay, in contrast with tissue culture assays, may not distinguish between viable and non-viable viruses. We evaluated, by comparison with tissue culture infectivity assay (plaque forming method), the ability of the gene probe assay to detect viable poliovirus 1 (LSc) in well water, autoclaved well water, filter-sterilized well water and autoclaved phosphate buffered saline kept at 37° C and 15° C for 75 days, and in dechlorinated tapwater held at room temperature. A gradual decline in numbers of poliovirus was observed in all of the samples by cell culture assay. With the exception of autoclaved well water and phosphate buffer samples, a parallel decline in virus detectable by gene probe occurred in all other water samples.

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