A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus

Table 1 Identification of neuraminidase subtypes of influenza A clinical nasopharyngeal aspirates.

Sample No. *	HA subtype^a	NA subtype	Subtype	Amplification by our RT-PCR	sequence
1	H3	NVI¹	H3	-	-
2	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
3	H3	NVI¹, N/A²	H3	+	N2
5	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	-
6	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
8	H3	N2	H3N2	+	N2
9	H3	NVI¹, N/A²	H3	-	-
10	H3	N2	H3N2	+	N2
12^e	H1	N1	H1N1	+	N2
14	H3	N2	H3N2 Wisconsin/67/2005 like strain	-	-
15	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
17	H3	NVI¹, N/A²	H3	+	NSA³
19	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
21	H3	NVI¹, N/A²	H3	+	N2
26	H3	N2	H3N2 Wisconsin/67/2005 like strain	-	-
27	H3	NVI¹	H3	-	-
30	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
31	H3	NVI¹	H3	+	NSA³
32	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
33	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
34	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
36	H3	NVI¹	H3	-	-
37	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
38	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
42	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
45	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
47	H3	NVI¹, N/A²	H3	-	-
50	H3	NVI¹, N/A²	H3	-	-
51	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
54	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
55	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
56	H3	NVI¹, N/A²	H3	-	-
57^c	NVI¹	NVI¹, N/A²	Not available	-	-
62^d	H5	N/A²	H5	+	N2
63^d	H5	N/A²	N/A²	-	-
64^d	Not H5	N/A²	N/A²	-	-
65	H3	N2	H3N2 Wisconsin/67/2005 like strain	+	N2
67^f	-	N/A²	Not available	+	NSA³

*Blinded specimens provided by Pathology Queensland. Only typed A influenza samples were listed. Type B influenza samples, adenovirus, and RSV samples were not included as they were all negative by our assay.
^a HA subtyping was performed by Queensland Forensic and Scientific Services using real-time PCR based on HA specific primers, as well as by hemaglutination and inhibition test to the viruses that were able to be cultured.
^c Sample 57 was not confirmed by a second laboratory for Influenza A infection nor was there virus isolated.
^d Samples 62, 63 and 64 are not clinical samples but are positive control RNA sample from WHO reference lab.
^e Sample was assayed and subtyped in duplicate to corroborate the NA subtype by sequence.
^f Sample 67 had pink contaminants floating in the RNA that could have affected PCR result.
NVI¹ = No virus isolated, therefore NA subtyping was unfeasible.
N/A² = Sample was not assayed with the corresponding test.
NSA³ = RT-PCR was positive but we were not able to obtain subtype by direct sequencing.

ISSN: 1743-422X