Diagnosis and quantitative evaluation of parvovirus B19 infections by real-time PCR in the clinical laboratory

Abstract

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 × 101 genome copies/reaction and to detect as few as 3 × 100 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 realtime PCR showed a mean of 1.1 × 109 B19 DNA copies/ml in samples in the acute active phase of B19 infection (DNA+, IgM+, IgG-), 4.3 × 106 B19 DNA copies/ml in samples in the active phase (DNA+, IgM+, IgG+), 3.7 × 105 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. © 2002 Wiley-Liss, Inc.