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 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.
Fini
F
F
Institute of Chemical Science, Division of Microbiology, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
Gallinella
G
G
Girotti
S
S
Zerbini
M
M
Musiani
M
M
D004279:DNA, Viral / Q000097:blood*; D006801:Humans; D008163:Luminescent Measurements; D016732:Parvovirus B19, Human / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity
D004279:DNA, Viral