Real-time quantitative PCR detection of four human bocaviruses

Abstract

Human bocavirus (HBoV) was discovered in 2005 and is associated with respiratory tract symptoms in young children. Three additional members of the genus Bocavirus, HBoV2, -3, and -4, were discovered recently from fecal specimens, and early results indicate an association between HBoV2 and gastrointestinal disease. In this study, we present an undifferentiating multiplex real-time quantitative PCR assay for the detection of these novel viruses. Differentiation of the individual bocavirus species can be subsequently achieved with corresponding singleplex PCRs or by sequencing. Both multiplex and singleplex assays were consistently able to detect ≤10 copies of HBoV1 to -4 plasmid templates/reaction, with dynamic quantification ranges of 8 logs and 97% to 102% average reaction efficiencies. These new assays were used to screen stool samples from 250 Finnish patients (median age, 40 years) that had been sent for diagnosis of gastrointestinal infection. Four patients (1.6%; median age, 1.1 years) were reproducibly positive for HBoV2, and one patient (0.4%; 18 years of age) was reproducibly positive for HBoV3. The viral DNA loads varied from <10(3) to 10(9) copies/ml of stool extract. None of the stool samples harbored HBoV1 or HBoV4. The highly conserved sequence of the hydrolysis probe used in this assay may provide a flexible future platform for the quantification of additional, hitherto-unknown human bocaviruses that might later be discovered. Our results support earlier findings that HBoV2 is a relatively common pathogen in the stools of diarrheic young children, yet does not often occur in the stools of adults.

FIG. 1.

Nucleotide sequence alignment of the HBoV1 to -4 genome segments used for qPCR. Intraspecies sequence variation is underlined and shown as degenerate bases according to IUPAC symbols (R, A+G; Y, C+T). Fully conserved nucleotides are shown without underlining, whereas nucleotide differences between the species are in bold. Arrow symbols show the positions and directions of the corresponding primers and probe. For HBoV2 and HBoV4, direct PCR product identification is possible by PCR product sequencing to distinguish three nucleotide differences (boxed nucleotides).

FIG. 2.

Representative results from the real-time PCR quantification of serial dilutions of HBoV1, HBoV2, HBoV3, and HBoV4 plasmids (10¹ to 10⁸ copies/reaction) with baseline-corrected fluorescence plotted against cycle number. Each type of plasmid was analyzed with the respective singleplex assay (solid curves) and the nondiscriminating multiplex assay (dotted curves). The insets show the corresponding standard curves with the logarithm of the input copy number (y) plotted against the corresponding C_q values (x), and the square of the correlation coefficient (R²). The standard curves for the singleplex (solid line) assays are almost indistinguishable from those of the multiplex (dotted line) assays.

FIG. 3.

qPCR quantification of 15 HBoV1 DNA positive sera with a previously established (1) reference assay (HR) plotted against the HBoV1 singleplex assay (HS) introduced in this study. r, Spearman's rank correlation coefficient.