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Sensitive and quantitative detection of PCR-amplified HIV-1 DNA products by an enzyme linked immunoassay following solution hybridization with two differently labelled oligonucleotide probes.


Böni, J; Schüpbach, J (1993). Sensitive and quantitative detection of PCR-amplified HIV-1 DNA products by an enzyme linked immunoassay following solution hybridization with two differently labelled oligonucleotide probes. Molecular and Cellular Probes, 7(5):361-371.

Abstract

We have developed and evaluated an ELISA-based detection method for PCR-amplified HIV-1 DNA. The assay uses two oligonucleotide probes which are end-labelled at the 5'-end with biotin or digoxigenin, respectively. Upon solution hybridization of these probes which react with the same strand of amplified DNA product, the formed hybrids are bound to avidin-coated wells of a microtitre plate and detected by horseradish peroxidase-labelled antibodies directed against digoxigen and 3, 3', 5, 5'-tetramethylbenzidine as substrate. Factors critical for a high signal-to-noise ratio were the use of serum as blocking agent, the amount of biotin-labelled and digoxigenin-labelled oligonucleotide probes present in a reaction and the inactivation of Taq DNA polymerase. The method has a detection limit of 1-3 pg of amplified DNA and is quantitative in a range extending from 1 pg to at least 200 pg. In the background of 1 microgram of total DNA, one single-stranded copy of HIV-1 DNA can be detected after 35 cycles of amplification. A comparison of the ELISA-based detection method with primer extension analysis, a method previously shown to reach a similar detection limit, demonstrated complete agreement of the results of 118 amplified DNAs. The method proved simple, requires only about 3 h, and could easily be adapted to the detection of other amplified target DNAs.

We have developed and evaluated an ELISA-based detection method for PCR-amplified HIV-1 DNA. The assay uses two oligonucleotide probes which are end-labelled at the 5'-end with biotin or digoxigenin, respectively. Upon solution hybridization of these probes which react with the same strand of amplified DNA product, the formed hybrids are bound to avidin-coated wells of a microtitre plate and detected by horseradish peroxidase-labelled antibodies directed against digoxigen and 3, 3', 5, 5'-tetramethylbenzidine as substrate. Factors critical for a high signal-to-noise ratio were the use of serum as blocking agent, the amount of biotin-labelled and digoxigenin-labelled oligonucleotide probes present in a reaction and the inactivation of Taq DNA polymerase. The method has a detection limit of 1-3 pg of amplified DNA and is quantitative in a range extending from 1 pg to at least 200 pg. In the background of 1 microgram of total DNA, one single-stranded copy of HIV-1 DNA can be detected after 35 cycles of amplification. A comparison of the ELISA-based detection method with primer extension analysis, a method previously shown to reach a similar detection limit, demonstrated complete agreement of the results of 118 amplified DNAs. The method proved simple, requires only about 3 h, and could easily be adapted to the detection of other amplified target DNAs.

Citations

15 citations in Web of Science®
15 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed
Communities & Collections:04 Faculty of Medicine > Institute of Medical Virology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:1 October 1993
Deposited On:11 Feb 2008 12:28
Last Modified:05 Apr 2016 12:22
Publisher:Elsevier
ISSN:0890-8508
Publisher DOI:10.1006/mcpr.1993.1054
PubMed ID:8264670

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