Development of real-time PCR assays for detection and quantification of human bocavirus

BACKGROUND: Human bocavirus (HBoV) is a parvovirus that has been recently detected in patients with respiratory illness. OBJECTIVES: We developed a sensitive, specific, and quantitative real-time PCR assay based on the TaqMan method for HBoV detection and quantification in respiratory specimens. STUDY DESIGN: Three individual real-time PCR assays were designed to amplify HBoV NS1, NP-1, and VP1 genes. For clinical evaluation, 506 nasal aspirates obtained from patients with acute respiratory tract infections during December 2006 to May 2007 were tested. RESULTS: Each assay had a broad dynamic range (50 × 10(7) to 5 × 10(7) copies of plasmid DNA) and high inter- and intra-assay reproducibility. The detection limit of each assay was 10 genome copies per reaction, and no crossreactivity with other major respiratory viruses or bacteria was detected. Clinical evaluation revealed that 11 (2.1%) of 506 patients diagnosed with upper respiratory tract infections, pneumonia, bronchitis, pharyngitis, or sinusitis had HBoV detected by all three assays, with viral loads ranging from 8.2 × 10(4) to 8.1 × 10(9) copies/ml of specimen. CONCLUSIONS: The three assays for HBoV diagnosis and quantification are highly sensitive, specific real-time tools for the reliable epidemiological and pathogenetic study of HBoV infection.