Simultaneous Differential Detection of H5, H7, H9 and Nine NA Subtypes of Avian Influenza Viruses via a GeXP Assay

Abstract

H5, H7 and H9 are the most important subtypes of avian influenza viruses (AIVs), and nine neuraminidase (NA) subtypes (N1–N9) of AIVs have been identified in poultry. A method that can simultaneously detect H5, H7, H9 and the nine NA subtypes of AIVs would save time and effort. In this study, 13 pairs of primers, including 12 pairs of subtype-specific primers for detecting particular subtypes (H5, H7, H9 and N1–N9) and one pair of universal primers for detecting all subtypes of AIVs, were designed and screened. The 13 pairs of primers were mixed in the same reaction, and the 13 target genes were simultaneously detected. A GeXP assay using all 13 pairs of primers to simultaneously detect H5, H7, H9 and the nine NA subtypes of AIVs was developed. The GeXP assay showed specific binding to the corresponding target genes for singlet and multiplex templates, and no cross-reactivity was observed between AIV subtypes and other related avian pathogens. Detection was observed even when only 102 copies of the 13 target genes were present. This study provides a high-throughput, rapid and labor-saving GeXP assay for the simultaneous rapid identification of three HA subtypes (H5, H7 and N9) and nine NA subtypes (N1–N9) of AIVs.

Simultaneous differential detection of H5, H7 and H9 subtypes of avian influenza viruses by a triplex fluorescence loop-mediated isothermal amplification assay

“…The swab specimens were directly eluted with 1 mL of PBS, and RNA was extracted from the centrifuged supernatant using the MagicPure ® Simple Viral DNA/RNA Kit (TransGen Biotech, Beijing, China). cDNA from different tissue samples of eight H5N2-infected SPF chickens was donated by the University of Connecticut, and cDNA from different tissue samples of three H7N9-infected SPF chickens was donated by China Agricultural University ( 19 ). The swab and tissue samples were evaluated by performing the TLAMP assay and previously published H5, H7, and H9 qPCR assays ( 16 ).…”