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

Alvarez AC, Brunck ME, Boyd V, Lai R, Virtue E, Chen W, Bletchly C, Heine HG, Barnard R - Virol. J. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochip Innovations Pty Ltd,, 8 Mile Plains, Queensland, Australia. alejandra.castillo@biochips.com.au

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.

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Related in: MedlinePlus

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).
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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).

Mentions: Bioinformatic analysis of each primer is presented in sequence logo format as shown in Figures 1, 2. The NA8F and NA10R primers were aligned against 3,337 sequences in the NCBI IVRD. When analyzing the last five bases at the 3'end of the primer NA8F, the alignment gave close to 100% match for all subtypes. In the case of the primer NA10R, the alignment gave close to 100% predicted match in the last 5 bases for all subtypes except for N2, N4, and N5 where it was 99.40%, 77% and 15% respectively. The N2 subtype has variability in the last five bases, but the frequency of variability is so low such that there is only one nucleotide difference in any single mismatched virus sequence. Despite the percentage of predicted mismatch in the last five 3' terminal bases of N4 and N5 subtypes they are detected with our primers. Thus, all 9 NA subtypes can be amplified as shown in Table 2 and Figure 3.


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

Alvarez AC, Brunck ME, Boyd V, Lai R, Virtue E, Chen W, Bletchly C, Heine HG, Barnard R - Virol. J. (2008)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483269&req=5

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).
Mentions: Bioinformatic analysis of each primer is presented in sequence logo format as shown in Figures 1, 2. The NA8F and NA10R primers were aligned against 3,337 sequences in the NCBI IVRD. When analyzing the last five bases at the 3'end of the primer NA8F, the alignment gave close to 100% match for all subtypes. In the case of the primer NA10R, the alignment gave close to 100% predicted match in the last 5 bases for all subtypes except for N2, N4, and N5 where it was 99.40%, 77% and 15% respectively. The N2 subtype has variability in the last five bases, but the frequency of variability is so low such that there is only one nucleotide difference in any single mismatched virus sequence. Despite the percentage of predicted mismatch in the last five 3' terminal bases of N4 and N5 subtypes they are detected with our primers. Thus, all 9 NA subtypes can be amplified as shown in Table 2 and Figure 3.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochip Innovations Pty Ltd,, 8 Mile Plains, Queensland, Australia. alejandra.castillo@biochips.com.au

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.

Show MeSH
Related in: MedlinePlus