Multiplex rRT-PCR for Respiratory Viruses

RNA was extracted from 140 µL of the collected samples using the QIAamp 96 Virus QIAcube HT kit (Qiagen, Manchester, UK), according to the manufacturer’s instructions. Samples were screened for 15 virus targets [26 ]—HRV, respiratory syncytial virus (A and B), human coronaviruses (OC43, NL63, and E229), influenza (A, B, and C), parainfluenza (1–4), adenovirus and human metapneumovirus—using in-house multiplexed real-time reverse transcription polymerase chain reaction (rRT-PCR) with a QuantiFast Multiplex RT-PCR kit (Qiagen, Manchester, UK) [28–30 (link)]. A sample was considered HRV positive if the rRT-PCR cycle threshold (Ct) value was <35.