Development of a chemiluminescence competitive PCR for the detection and quantification of parvovirus B19 DNA using a microplate luminometer.

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.

journal_name

Clin Chem

journal_title

Clinical chemistry

authors

Fini F,Gallinella G,Girotti S,Zerbini M,Musiani M

subject

Has Abstract

pub_date

1999-09-01 00:00:00

pages

1391-6

issue

9

eissn

0009-9147

issn

1530-8561

journal_volume

45

pub_type

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