Sample preparation

Nasopharyngeal aspirate specimens are placed in 1mL of sterile saline and vigorously mixed on a Vortex. Nucleic acids are extracted from 0.2 mL of this suspension using the Roche MagNAPure™ instrument (Roche Diagnostics, Australia) as per manufacturer's instructions.

Influenza A oligonucleotides (targeting the influenza A matrix gene) Forward CTTCTAACCGAGGTCGAAACGTAa Reverse GGTGACAGGATTGGTCTTGTCTTTAa Probe Fam-TCAGGCCCCCTCAAAGCCGAG-BHQ1a

Reaction mix (Qiagen One-Step RT-PCR Kit; Qiagen, Australia)

Qiagen One-Step RT-PCR Enzyme mix:

1.0

|L

Qiagen One-Step RT-PCR Buffer (5x):

5.0

|L

Qiagen One-Step RT-PCR dNTP mix

(10 mM):

1.0

|L

Forward primer (10 |M):

2.0

|L

Reverse primer (10 |M):

2.0

|L

Probe (20 |jM):

0.2

|L

RNase-free water:

8.8

|L

Specimen or control nucleic acid extract:

5.0

|L

Total:

25.0

|L

Cycling conditions (ABI Prism® 7500; Applied

Biosystems, USA)

RT step 1 cycle:

50°C 20 min Activation/denaturation 1 cycle:

95°C 15 min Cycling 45cycles:

95°C 15 sec 60°C 60 sec acquisition

Result interpretation: Specimens providing exponential amplification curves are considered positive for influenza A RNA.

a These sequences matched the matrix protein genes of a broad range on influenza A subtypes, including H1N1 (Genbank accession number AY619976), H1N2 (AY233392), H2N2 (M63531), H3N2 (AY210264), H5N1 (AY646180), H5N3 (AY300973), H7N2 (AY241624), H7N3 (AY611525), H7N7 (AJ619676), H9N2 (AF523497).

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