HRSV Subtype Identification by RT-PCR

The viral nucleic acid was directly extracted from the clinical specimens using the Tianlong nucleic acid extraction kit (Tianlong Biotechnology, Xian, China) according to the manufacturer’s instructions. The samples were screened for human respiratory pathogens, including 16 viruses and 11 bacteria using multiplex real-time reverse transcriptase PCR (RT-PCR) with the TaqMan low-density array (TLDA) kit (Thermo Fisher Scientific Inc., Waltham, USA). The subtypes of HRSV were further identified by in-house real-time RT-PCR (20 (link)). The second hypervariable region (HVR2) of the G gene of HRSV B subtype was amplified using a one-step reverse transcription-PCR kit (TaKaRa Biotechnology, Dalian, China) and the primer pair GPB/F1 (21 (link)). The reaction conditions, as well as the purification and sequencing protocols, were as described previously (21 (link), 22 (link)).