A real-time PCR assay was developed for quantitative detection of B19
DNA in clinical serum samples. The assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent
double-stranded DNA binding
dye SYBR Green I. With an optimized PCR protocol, this system was able to quantitate the target
DNA down to 3 x 10(1) genome copies/reaction and to detect as few as 3 x 10(0) genome copies/reaction. Real-time PCR was used to detect B19
DNA in 108 serum samples from patients with a clinical suspicion of B19
infection, showing a sensitivity of 92.7% and a specificity of 100% when compared with a standardized PCR-ELISA considered as the standard. Using the LightCycler assay, the entire procedure of detection and quantitation of B19
DNA in clinical serum samples took up to 90 min proving five times faster than PCR-ELISA. B19
DNA quantitation in positive samples by real-time PCR showed a mean of 1.1 x 10(9) B19
DNA copies/ml in samples in the acute active phase of B19
infection (
DNA+,
IgM+,
IgG-), 4.3 x 10(6) B19
DNA copies/ml in samples in the active phase (
DNA+,
IgM+,
IgG+), 3.7 x 10(5) genome copies/ml in samples in the long-lasting active phase (
DNA+,
IgM-,
IgG+) with a statistically significant reduction of B19
DNA content between the group of sera in the acute active phase and the group of sera in the active phase of B19
infection. The high levels of sensitivity, specificity, and rapidity provided by the LightCycler technology for the detection and quantitation of B19
DNA represent a significant improvement for the laboratory diagnosis of B19
infection.