Real-Time Quantitative PCR Detection of Four Human Bocaviruses (cid:1)

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 corre- sponding 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. extracts was 40 ng/ (cid:2) l (25th percentile, 20 ng/ (cid:2) l; 75th percentile, 77 ng/ (cid:2) l; range, 2 to 200 ng/ (cid:2) l). Potential PCR inhibition had been studied with 80 of the 250 DNA extracts by DNA spiking (17). None of the spiked samples were inhibitory. To further evaluate our qPCR assay, we analyzed three stool DNA extracts shown by qualitative PCR to contain HBoV2, HBoV3, or HBoV4 DNA (12). Plasmids. The performances of the qPCR assays were studied with plasmids containing the joint region of the left-hand untranslated region (UTR) and the NS1 gene of the human bocavirus 1 to 4 genomes, corresponding in HBoV1 to nucleotides (nt) 98 to 388 (GenBank accession number EU984245). The corre- sponding regions of HBoV2 (FJ170279) and HBoV3 (EU918736) were synthesized and cloned into pUC57 by Genscript (NJ), and that of HBoV4 was syn- thesized and cloned into pJet1.2 (Fermentas, Burlington, Canada) in Helsinki, Finland. The near-full-length HBoV1 clone, pST2, was a kind gift from Tobias Allander (2). Plasmid DNA concentrations were determined by an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). Each plasmid was diluted serially from 0.5 (cid:3) 10 8 to 0.5 (cid:3) 10 1 copies/ (cid:2) l in 10 mM Tris-EDTA buffer, aliquoted, and at (cid:4) 20°C until use for generation of standard curves for DNA quantiﬁcation. genotypes 1 (AY504945), 2 (AY044266) and 3 (AJ249437; a kind gift from Antoine Garbarg-Chenon), and polyomavi- ruses BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40) (kind gifts from Kristina Do¨rries). We also tested a partial genome of human parvovirus 4 (PARV4) and the late-region genes of KI polyomavirus (KIPyV) (EF127906; a kind gift from Tobias Allander). We further tested pooled nucleic acid extracts of human stool containing astrovirus, norovirus, and rotavirus. The stool specimens had been found virus positive by diagnostic electron microscopy and/or PCR by the Helsinki University Central Hospital Laboratory Division.