Human Bocavirus in Patients with Respiratory Tract Infection

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

Total nucleic acid was extracted from 100 µL of the sample by using Instagene Matrix Kit (Bio-Rad, Hercules, CA, USA). PCR primers were designed using Beacon Designer software (Premier Biosoft International, Palo Alto, CA, USA) in the conserved region of the NS-1 coding region of the HBoV genome by using the HBoV ST2 sequence (Genbank accession number: DQ000496). The forward primer (5´-GCAAATCTCTTCTGGCTACACG-3´) and the reverse primer (5´-CCTCTGCGATCTCTATATTGAAGG-3´) were targeted at a portion of the HBoV NS-1 gene. The conventional PCR reaction mixture consisted of 0.2 pg/μL forward and reverse primers, 2.5 mM dNTPs, 50 mM KCl, 1.5 mM MgCl2, 5 U of Taq polymerase, and 3 μL of extracted DNA in a final volume of 25 μL. The PCR cycling conditions consisted of 35 cycles (involving reaction at 30 sec at 94˚C, 30 sec at 60˚C, and 30 sec at 72˚C) after the preheating step of 3 min at 94˚C. All the PCR products obtained from positive reactions were sequenced completely to confirm sequence specificity.