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

Background: Quantitative PCR of viral nucleic acids can be useful clinicall y in diagnosis, risk assessment, and monitoring of antiviral therapy. We wi shed to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B 19. Methods: Parvovirus DNA target sequences and competitor sequences were coam plified and directly labeled. Amplified products were then separately hybri dized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively anal yzed by a microplate luminometer and were correlated to the amounts of ampl ified 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 low er (20 genome copies) than colorimetric substrates (50 genome copies). In c hemiluminescence 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 contr ol sera. Conclusions: The chemiluminescence cPCR appears to be a sensitive and speci fic method for the quantitative detection of viral DNAs. (C) 1999 American Association for Clinical Chemistry.