Multiplex qRT-PCR Assay for Respiratory Viruses

The specimens were vortexed, and a 100-µL volume was used for total nucleic acid extraction using the QIAamp Viral RNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. One step qRT-PCR was performed by using the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, Carlsbad, California, USA). NP and OP specimens from each patient were separately tested by singleplex qRT-PCR for eight viral pathogens: adenovirus, influenza A virus, influenza B virus, human metapneumovirus (hMPV), parainfluenza viruses (PIV) 1–3, and respiratory syncytial virus (RSV). The primers, probes and positive controls for all viruses were provided by CDC-Atlanta. Sequences for the primers and probes are shown in Table 2[11] . We tested for influenza A virus with the conserved matrix gene-base qRT-PCR; positive influenza A samples were also subtyped as 2009 pandemic influenza A (H1N1) virus (2009 H1N1), influenza A (H3N2) virus (H3N2), and seasonal influenza A (H1N1) virus (H1N1) [12] . Fluorescence was read at the combined annealing-extension step at 57°C and recorded as threshold cycle (C_t) values. A C_t value ≤39.9 was regarded as positive; C_t values ≥40.0 were regarded as negative. The qRT-PCR test did not discriminate between viral mRNA and genomic RNA. Specimens were not tested if the following conditions existed when the specimen arrived at the lab: there was no swab, the volume was less than 600 µL, the specimen was at room temperature, patient identification was absent or inadequate, or the patient questionnaire was absent. In addition, the test results were discarded for any specimen whose internal control (human ribonuclease P gene) was negative.