Nucleic acids were extracted from 200 μL of NPA by using the QIAamp viral RNA Mini Kit (QIAGEN, Inc., Mississauga, Ontario, Canada). A duplex HBoV PCR (TaqMan assay) was used to amplify conserved regions of NP-1 and NS-1 genes as described (14) , except that the NS-1 forward primer was replaced with primer 5′-TAG TTG TTT GGT GGG ARG A-3′. Probes were labeled with 6-carboxyfl uorescein (FAM) or tetrachloro-6-carboxyfl uorescein (TET) at the 5′ end and with a quencher at the 3′ end. Amplicons were 81 bp (NP-1) and 74 bp (NS-1), respectively. Duplex amplifi cation was conducted by using 1 μmol/L NS-1 forward primer and 0.4 μmol/L NS-1 reverse primer and the 2 NP-1 primers. Taqman probes were used at concentrations of 0.1 mmol/L for the NP-1 gene and 0.2 mmol/L for the NS-1 gene (14) . The amplifi cation master mixture consisted of 2.5 mmol/L MgCl 2 , 3.33 mg/mL bovine serum albumin, 0.2 mmol/L of each of the 4 deoxynucleotide triphosphates (Amersham Biosciences, Uppsala, Sweden), 10 mmol/L Tris-HCl, 50 mmol/L KCl, 0.625 U Promega Taq DNA polymerase (Fisher Scientifi c, Markham, Ontario, Canada) combined with TaqStart antibody (BD Biosciences Clontech, Palo Alto, CA, USA), and 3 μL DNA in a fi nal volume of 25 μL. PCR amplifi cation (180 s at 94°C and 45 cycles for 10 s at 95°C, 30 s at 58°C, and 30 s at 72°C) was performed in a Smart Cycler thermal cycler (Cepheid, Sunnyvale, CA, USA). A PCR extension step of 5 min at 72°C was performed at the end of the cycling protocol. An HBoV infection was defi ned by a positive PCR result for NP-1 and NS-1. The duplex assay had a sensitivity of 10 genome copies for NP-1 and NS-1 gene targets on the basis of quantifi cation analysis of positive control plasmids.