Development of a Chemiluminescence Competitive PCR for the Detection and Quantification of Parvovirus B19 DNA Using a Microplate Luminometer

Development of a Chemiluminescence Competitive PCR for the Detection and Quantification of... Abstract Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. Methods: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products. Results: Luminol-based systems displayed constant emission but had a higher detection limit (100–1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera. Conclusions: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs. © 1999 The American Association for Clinical Chemistry « Previous | Next Article » Table of Contents This Article Clinical Chemistry September 1999 vol. 45 no. 9 1391-1396 » Abstract Full Text PDF Classifications Molecular Diagnostics and Genetics Services Email this article to a friend Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Download to citation manager Responses No responses published Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Fini, F. Articles by Musiani, M. Search for related content PubMed PubMed citation Articles by Fini, F. Articles by Musiani, M. Related Collections Molecular Diagnostics and Genetics Related Content Load related web page information Follow Us Clinical Chemistry Trainee Council Register Today! www.traineecouncil.org Information for Authors Submit a Manuscript Editorial Board Clinical Case Studies Clinical Chemistry Guide to Scientific Writing Journal Club Podcasts Translated Content Annual Meeting Abstracts Permissions and Reprints Advertising Copyright © 2012 by the American Association for Clinical Chemistry http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Chemistry American Association for Clinical Chemistry

Development of a Chemiluminescence Competitive PCR for the Detection and Quantification of Parvovirus B19 DNA Using a Microplate Luminometer

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Abstract

Abstract Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. Methods: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products. Results: Luminol-based systems displayed constant emission but had a higher detection limit (100–1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera. Conclusions: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs. © 1999 The American Association for Clinical Chemistry « Previous | Next Article » Table of Contents This Article Clinical Chemistry September 1999 vol. 45 no. 9 1391-1396 » Abstract Full Text PDF Classifications Molecular Diagnostics and Genetics Services Email this article to a friend Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Download to citation manager Responses No responses published Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Fini, F. Articles by Musiani, M. Search for related content PubMed PubMed citation Articles by Fini, F. Articles by Musiani, M. Related Collections Molecular Diagnostics and Genetics Related Content Load related web page information Follow Us Clinical Chemistry Trainee Council Register Today! www.traineecouncil.org Information for Authors Submit a Manuscript Editorial Board Clinical Case Studies Clinical Chemistry Guide to Scientific Writing Journal Club Podcasts Translated Content Annual Meeting Abstracts Permissions and Reprints Advertising Copyright © 2012 by the American Association for Clinical Chemistry

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Clinical ChemistryAmerican Association for Clinical Chemistry

Published: Sep 1, 1999

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