SUPPLEMENTAL DIGITAL CONTENT 1
PCR for respiratory viruses methodology
Total nucleic acid was recovered from 200µL aliquots of swab material in
STGG broth using a Kingfisher 96 magnetic particle processor (Thermo Scientific) and
eluted in 60µL sterile water. Viral DNA was amplified using real time PCR assays, and
viral RNA using two-step reverse transcription-real time PCR assays. Complementary
DNA (cDNA) synthesis was performed in 25µL volumes using 100 units MMLV
reverse transcriptase (Promega) and 0.5mg/mL random hexamers for 30 minutes at
37°C followed by 10 minutes at 95°C. Real time PCR was performed in 20µL reactions
consisting of ABI Fast Universal Master Mix (Applied Biosystems) with 5µL total
nucleic acid extract or cDNA and primers and probes to detect influenza A (IFA) and B
(IFB) viruses1, respiratory syncytial virus types A and B (RSV) (SDC 2), human
metapneumoviruses (HMPV)2, parainfluenzavirus (PIV) types 1-3 (SDC 2), human
rhinoviruses (RV)3, enteroviruses (EV)4, human adenoviruses (ADV)5, and human
bocavirus (HBoV)6. A two temperature thermal cycling protocol (50 cycles of 95°C
denaturation and 60°C annealing/extension) in an ABI 7500 Fast processor (Applied
Biosystems) was used.
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