Human Bocavirus in Patients with Respiratory Tract Infection

Kim, Jang Su; Lim, Chae Seung; Kim, Young Kee; Lee, Kap No; Lee, Chang Kyu

Real-time PCR for viral load was performed on the specimens positive for HBoV by conventional PCR. A TaqMan probe (5´-ATGTTGCCGCCAGTAACTCCACCC-3´) was labeled at the 5´ ends with the reporter molecule FAM and at the 3´ ends with Black Hole Quencher 1 (Biosearch Technologies, Inc., Novato, CA, USA). The assay was performed using Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia) and the standard protocol of TaqMan universal PCR master mix (Applied Biosystems, Foster City, CA, USA); each 25 µL sample of the reaction mixture contained 10 pg/ µL of the forward and the reverse primers and 3 µL of the extracted DNA. Amplification conditions consisted of reactions for 3 min at 50˚C, 3 min at 94˚C, and 40 cycles of 30 sec at 94˚C, 30 sec at 60˚C and 30 sec at 72˚C maintained for 3 min. Detection limit of real-time PCR for HBoV was 1.3 × 10 3 copies/mL, which corresponded to 33 copies per reaction.