Real-time PCR assays for detection of bocavirus in human specimens - PubMed (original) (raw)
Real-time PCR assays for detection of bocavirus in human specimens
Xiaoyan Lu et al. J Clin Microbiol. 2006 Sep.
Abstract
The recently discovered human bocavirus (HBoV) is the first member of the family Parvoviridae, genus Bocavirus, to be potentially associated with human disease. Several studies have identified HBoV in respiratory specimens from children with acute respiratory disease, but the full spectrum of clinical disease and the epidemiology of HBoV infection remain unclear. The availability of rapid and reliable molecular diagnostics would therefore aid future studies of this novel virus. To address this, we developed two sensitive and specific real-time TaqMan PCR assays that target the HBoV NS1 and NP-1 genes. Both assays could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV genome, with a dynamic range of 8 log units (10(1) to 10(8) copies). Eight blinded clinical specimen extracts positive for HBoV by an independent PCR assay were positive by both real-time assays. Among 1,178 NP swabs collected from hospitalized pneumonia patients in Sa Kaeo Province, Thailand, 53 (4.5%) were reproducibly positive for HBoV by one or both targets. Our data confirm the possible association of HBoV infection with pneumonia and demonstrate the utility of these real-time PCR assays for HBoV detection.
Figures
FIG. 1.
NS1 and NP-1 real-time PCR assays showing representative results obtained with serial 10-fold dilutions (101 to 108 copies per reaction) of the pCRII HBoV plasmid. The top panels show baseline subtractive curve fit views of the data with relative fluorescence units (RFU) plotted against cycle number. The default setting of 10 times the standard deviation of the RFU measured in all wells over the baseline cycles was used to calculate the CT for a positive reaction (horizontal line). The bottom panels show standard curve analysis of the DNA amplification plots with CT values plotted proportionately against the logarithm of the input copy number. The dynamic range of NS1 and NP-1 PCR assays spans 8 log units with slopes of −3.3 and an R 2 value of 1.0.
FIG. 2.
CT values of NS1 plotted against NP-1 for 48 clinical specimens positive for both targets.
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