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

Abstract

Background: The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes.

Results: Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y.

Conclusion: Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

Figure 1

Nucleotide sequence alignment of NA8F primer. A sequence logo representation of 3,337 available sequences in the NCBI database at the time the study was conducted. All 9 NA subtypes were aligned against the NA8F primer and analyzed for discrepancies at the 3'end. The big letters represent the consensus sequences for each subtype. The standard mixed base definition was applied, and for reference "I" stands for inosine. ^aAlignment is presented in sequence logo format [32-34].

Figure 2

Nucleotide sequence alignment of NA10R primer. A sequence logo representation of 3,337 available sequences in the NCBI database at the time the study was conducted. All 9 NA subtypes were aligned against the NA10R primer and analyzed for discrepancies at the 3'end. The big letters represent the general consensus sequences for each subtype and at the end a general consensus sequence is given for the total population of samples. The standard mixed base definition was applied, and for reference "I" stands for inosine. ^aAlignment is presented in sequence logo format [32-34].

Figure 3

One-step RT-PCR amplification of NA gene from all 9 NA subtypes using animal samples from allantoic fluids. A fragment of approximately 253 bp was amplified using primers containing M13 sequence. Example of some subtypes (refer to Table 2 for strain names) assayed: M, 100 bp DNA Ladder (Promega); 1) H9N2, 2) H16N3, 3) H8N4, 4) H14N5, 5) H13N6, 6) H10N7, 7) H11N9, 8) H5N1, 9) negative control (water instead of template), 10) H6N5, 11) H13N6, 12) H14N6, 13) H7N7, 14) H3N8, 15) H11N9, 16) negative control (water instead of template).

Figure 4

One-step RT-PCR amplification of NA gene from clinical NPA samples. Example of amplification results of Influenza A and influenza B. Sample numbers correspond to the samples described in Table 1, except sample 35 which is influenza B (description data not shown); M, 100 bp DNA Hyperladder II (Bioline); and negative control (- ctrl: water instead of template).

Figure 5

Sensitivity of the one-step RT-PCR assay. Example of amplification results of ten-fold serial dilutions of in-vitro transcribed RNA H10N7 subtype (refer to Table 2 for strain name). A band of approximately 253 bp was clearly visible with 40 femtogram of starting RNA (equivalent to 10⁵ copies). M, 100 bp DNA Hyperladder II (Bioline); and negative control (- ctrl: water instead of template).