ELECTRONIC SUPPLMENTARY MATERIAL
Appendix 1
Genetic Analysis Methods
Virus RNA extraction
After initial storage at -80oC, NPA samples were thawed, vortexed and centrifuged at 1000 x g for 3 minutes at 4C to remove debris. Swab tip extracts were prepared by immersing the tips in Buffer AVL (Qiagen, Valencia, CA) and thoroughly vortexing. Virus RNA was then extracted from 280 µl of the clarified NPA supernatants or swab tip extracts using the QIAamp Virus RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was eluted from the QIAamp spin columns in 60 µl Buffer AVE (supplied) and stored at -80C.
DNA extraction
DNA was extracted from NPA samples that had sufficient volume remaining following RNA extraction. Total DNA was extracted from 200 µl of the clarified NPA supernatant using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. DNA was eluted from the QIAamp spin columns in 60 µl Buffer AE (supplied) and stored at -20C.
Hexaplex assay
Multiplex reverse transcription-polymerase chain reaction-enzyme hybridization assay (RT-PCR-EHA) (Prodesse, Waukesha, WI) was carried out according to the manufacturer’s instructions. Briefly, RNA was reverse transcribed to cDNA using RT Mix (Prodesse) and MuLV RT enzyme (Applied Biosystems, Foster City, CA) in the presence of RNase inhibitor (Applied Biosystems). PCR was performed using AmpliTaq Gold DNA polymerase (Applied Biosystems) and Hexaplex Super-Mix (Prodesse), which contains 6.5 pairs of biotinylated primers designed to hybridize to Inf A and B, para 1, 2 and 3 and RSV types A and B. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and heat denatured. The denatured amplification products were added to the supplied streptavidin-coated 96-well plates together with hybridization solutions containing peroxidase-labelled probes that targeted each of the seven viruses. After capture and hybridization reaction, the wells were washed six times each in 300 µl of Wash Solution (Prodesse) before addition of substrate solution for 10 minutes. The reaction was terminated with Stop Solution (Prodesse) and the optical density of each well measured at 450 nm using a Multiskan EX microplate photometer (Thermo Electron Corporation, Waltham, MA, USA).
A positive result was defined as an OD ≥ 0.400, with a reading at least 4 times greater than the negative control. An internal positive control was included for every sample to monitor the presence of PCR inhibitors. The RSV A probe detects both RSV A and RSV B, whilst the RSV B probe will bind only to RSV B. Hence, for samples positive for RSV B, it is not possible to discount a dual RSV A and RSV B infection.
Real-time PCR
The respiratory sample cDNA was analyzed for the presence of human EV, hMPV, RV and CoV (non-SARS) by real-time RT-PCR using an ABI PRISM 7700 Sequence Detection system (Applied Biosystems). Previously published assays were utilized for the detection of CoV9 (A1), hMPV (A2) and EV (A3). An established in-house assay was used for the detection of RV, where primers and probe target the 5’ untranslated region (UTR) of RV (Table A1). All amplifications were carried out in a 25 µl reaction mixture of 5 µl cDNA, primers and probes and a commercial polymerase solution (RealMasterMix Probe; Eppendorf AG, Hamburg, Germany) using the following conditions: an initial cycle of 1 minute 30 seconds at 95C, followed by 40 cycles of 15 seconds at 95C and 1 minute at 60C. DNA extracted from NPA samples was analyzed for the presence of AV by real-time PCR using a previously described protocol (A4).
All probes for real-time PCR were labeled with the 5’ reporter dye 6-carboxy-fluorescein (FAM), and the 3’ quencher dye 6-carboxy-tetramethyl-rhodamine (TAMRA). No-template controls were included in every experiment to monitor reagent contamination. Positive controls for real-time PCR were RV serotype 16 (American Type Culture Collection (ATCC), Manassas, VA, USA), EV coxsackievirus type A (a gift of Dr Darren Shafren, University of Newcastle, Australia), hHMPV (clinical isolate, 2001, Newcastle, Australia) and AV type 1 (ATCC).
Rhinovirus primer/probe / Sequence, 5’ 3’ / Final concentration (nM)Probe / FAM-AGSCTGCGTGGCKGCCWR-TAMRA / 200
Fwd primer / ATTCCCCACTGGCGACAG / 900
Rev primer / AGGGTTAAGGTTAGCCACATTCAG / 900
Table A1. Oligonucleotide primer and probe sequences for real-time RT-PCR of human rhinovirus. Degenerate bases: R = G or A; W = T or A; S = C or G; K = G or T.
Appendix references
A1van Elden LJ, van Loon AM, van Alphen F, et al. Frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction. Journal of Infectious Diseases;189(4):652-7.
A2. Maertzdorf J, Wang CK, Brown JB, et al. Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages. J Clin Micro 2004;42(3):981-6.
A3. Nijhuis M, van Maarseveen N, Schuurman R, et al. Rapid and
sensitive routine detection of all members of the genus
enterovirus in different clinical specimens by real-time PCR.
J Clin Micro. 40(10) 2002;40(10):3666-70.
A4Heim A, Ebnet C, Harste G, Pring-Akerblom P. (2003) Rapid
and quantitative detection of human adenovirus DNA by real-
time PCR. J Med Virol70: 228-239.