tetano
Editor, Senior Moderator
Influenza Other Respir Viruses. 2015 Dec 17. doi: 10.1111/irv.12370. [Epub ahead of print]
[h=1]Simultaneous detection of four different neuraminidase types of avian influenza A H5 viruses by multiplex reverse transcription-PCR using a GeXP analyser.[/h] Li M[SUP]1[/SUP], Xie Z[SUP]1[/SUP], Xie Z[SUP]1[/SUP], Liu J[SUP]1[/SUP], Xie L[SUP]1[/SUP], Deng X[SUP]1[/SUP], Luo S[SUP]1[/SUP], Fan Q[SUP]1[/SUP], Huang L[SUP]1[/SUP], Huang J[SUP]1[/SUP], Zhang Y[SUP]1[/SUP], Zen T[SUP]1[/SUP], Feng J[SUP]2[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]BACKGROUND:[/h] Highly pathogenic avian influenza (HPAI) H5-subtype viruses such as H5N1, H5N2, H5N6 and H5N8 cause considerable economic damage to the global poultry industry and pose a major public health hazard worldwide. Thus, rapid identification of avian influenza virus (AIV)-infected poultry is essential for the effective control of H5-subtype AIVs.
[h=4]METHODS:[/h] Six pairs of primers were designed using conserved and specific sequences of the AIV subtypes H5, N1, N2, N6 and N8 in GenBank, and single and mixed avian pathogen cDNA/DNA templates were employed to evaluate the specificity of a multiplex assay with a GeXP analyser. Corresponding specific DNA products were amplified for each gene, revealing amplification peaks for M, H5, N1, N2, N6 and N8 genes from four different NA subtypes of influenza A H5 virus. The GeXP assay was further evaluated using 180 clinical specimens and compared with RRT-PCR (real-time reverse transcriptase-PCR) and virus isolation.
[h=4]RESULTS:[/h] The multiplex PCR assay demonstrated excellent specificity, with each pair of specific primers generating only products corresponding to the target genes and without cross-amplification with other NA-subtype influenza viruses or other avian pathogens. Using various premixed ssRNAs containing known AIV target genes, the detection limit for the multiplex assay was determined to be 10[SUP]2[/SUP] copies/μl. Analysis of 180 clinical samples using the GeXP assay revealed detection comparable to that using RRT-PCR and virus isolation.
[h=4]CONCLUSIONS:[/h] This GeXP analyser-based multiplex assay for four different NA subtypes of H5 HPAI viruses is both highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[h=4]KEYWORDS:[/h] GeXP analyser; H5 avian influenza viruses; Multiplex detection; NA typing
PMID: 26677838 [PubMed - as supplied by publisher] Free full text
[h=1]Simultaneous detection of four different neuraminidase types of avian influenza A H5 viruses by multiplex reverse transcription-PCR using a GeXP analyser.[/h] Li M[SUP]1[/SUP], Xie Z[SUP]1[/SUP], Xie Z[SUP]1[/SUP], Liu J[SUP]1[/SUP], Xie L[SUP]1[/SUP], Deng X[SUP]1[/SUP], Luo S[SUP]1[/SUP], Fan Q[SUP]1[/SUP], Huang L[SUP]1[/SUP], Huang J[SUP]1[/SUP], Zhang Y[SUP]1[/SUP], Zen T[SUP]1[/SUP], Feng J[SUP]2[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]BACKGROUND:[/h] Highly pathogenic avian influenza (HPAI) H5-subtype viruses such as H5N1, H5N2, H5N6 and H5N8 cause considerable economic damage to the global poultry industry and pose a major public health hazard worldwide. Thus, rapid identification of avian influenza virus (AIV)-infected poultry is essential for the effective control of H5-subtype AIVs.
[h=4]METHODS:[/h] Six pairs of primers were designed using conserved and specific sequences of the AIV subtypes H5, N1, N2, N6 and N8 in GenBank, and single and mixed avian pathogen cDNA/DNA templates were employed to evaluate the specificity of a multiplex assay with a GeXP analyser. Corresponding specific DNA products were amplified for each gene, revealing amplification peaks for M, H5, N1, N2, N6 and N8 genes from four different NA subtypes of influenza A H5 virus. The GeXP assay was further evaluated using 180 clinical specimens and compared with RRT-PCR (real-time reverse transcriptase-PCR) and virus isolation.
[h=4]RESULTS:[/h] The multiplex PCR assay demonstrated excellent specificity, with each pair of specific primers generating only products corresponding to the target genes and without cross-amplification with other NA-subtype influenza viruses or other avian pathogens. Using various premixed ssRNAs containing known AIV target genes, the detection limit for the multiplex assay was determined to be 10[SUP]2[/SUP] copies/μl. Analysis of 180 clinical samples using the GeXP assay revealed detection comparable to that using RRT-PCR and virus isolation.
[h=4]CONCLUSIONS:[/h] This GeXP analyser-based multiplex assay for four different NA subtypes of H5 HPAI viruses is both highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[h=4]KEYWORDS:[/h] GeXP analyser; H5 avian influenza viruses; Multiplex detection; NA typing
PMID: 26677838 [PubMed - as supplied by publisher] Free full text