PCR amplification of bocaviruses. We used nested PCR targeting the VP1/2 region of both HBoV1 and HBoV2 (nucleotide positions 3233-3808, numbered here and subsequently with use of the HBoV2 prototype sequence; GenBank accession number FJ170278). Nucleic acids (both DNA and RNA) were extracted (QIAamp Viral RNA mini kit) from 140 mL of clarified stool supernatant and were eluted into 60 mL of water. First-round PCR primers were AK-VP-F1 (5 -CGCCGTGGCT-CCTGCTCT-3 ) and AK-VP-R1 (5 -TGTTCGCCATCACAAA-AGATGTG-3 ) and second-round primers were AK-VP-F2 (5 -GGCTCCTGCTCTAGGAAATAAAGAG-3 ) and AK-VP-R2 (5 -CCTGCTGTTAGGTCGTTGTTGTATGT-3 ). PCR reactions contained 2.5 U of Taq DNA polymerase (NEB) in 1.1ϫ Thermopol reaction buffer with MgCl (2.0 mmol/L), 20 pmol/ L (each) of forward and reverse primers, and 2.5 mL of nucleic acids (first round) or 1 mL of the first-round PCR product (second round) as template in a 50 mL total volume. Firstround conditions were 10 cycles at 95ЊC for 35 s, 58ЊC for 1 min, and 72ЊC for 1 min, with a decrease of 0.5ЊC in annealing temperature each cycle; 30 cycles at 95ЊC for 30 s, 54ЊC for 45 s, and 72ЊC for 45 s; and a final extension at 72ЊC for 10 min. Similar conditions were used for the second PCR round, except that the initial annealing temperatures were 60ЊC and 58ЊC in the first and second group of PCR cycles, respectively. Amplicons of the appropriate size, as detected by agarose gel electrophoresis, were directly sequenced. The PCR products whose sequences are reported here produced unambiguous dideoxysequencing electrophoregram peaks indicating the predominance (190%) of a single bocavirus variant. The sensitivity of the pan-bocavirus nested PCR was determined using dilution of plasmids containing the target sequences from each of the 4 bocavirus species and was estimated at 10-100 plasmid copies for each species.