Distinctive clinical features of human bocavirus in children younger than 2 years

Moriyama, Yoko; Hamada, Hiromichi; Okada, Mineyuki; Tsuchiya, Nozomi; Maru, Hiromi; Shirato, Yuri; Maeda, Yasuhiro; Hirose, Yosuke; Yoshida, Masaki; Omura, Yoh; Honda, Takafumi; Muto, Ayako; Hayashi, Kitami; Terai, Masaru

A nasopharyngeal swab was collected from each patient and then dipped into saline and stored at −80°C until nucleic acid extraction. Nucleic acid was extracted using a kit (High Pure Viral Nucleic Acid Kit; Roche Diagnostics Corp., Mannheim, Germany) according to the manufacturer's recommended procedures. Then PCR/reverse-transcription PCR (RT-PCR) was performed using previously described methods with slight modifications [1, 3, 4, 9, 22, 24] . Briefly, for the RNA viruses, a superscript one-step RT-PCR system (Invitrogen Corp., Carlsbad, CA, USA) was used for reverse-transcription and first-round PCR with virus specific primers under the manufacturer's instructions. For DNA viruses and also nested PCR, Ex Taq DNA polymerase (Takara Bio Inc., Shiga, Japan) was used along with the manufacturer's supplied buffer under the appropriate thermal cycle conditions for each primer. Each sample was examined for RSV, rhinovirus (RV), hMPV, HBoV, adenovirus (AdV), and parainfluenzavirus (PIV). Following amplification, PCR products were separated on the 1.5% agarose gel with Tris-boric acid-EDTA buffer and visualized with SYBR-green (Cambrex Corp., Rockland, ME, USA) under a UV transilluminator. The PCR products were purified; their nucleic acid sequences were determined to confirm PCR results. Nucleotide sequences were processed using software (GENETYX) and compared to nucleotide sequences of the DNA database using a BLAST system [20].