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. 2020 Jan 15;12(1):100. doi: 10.3390/v12010100

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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The one-step multiplex RT-qPCR assay for the detection of pan-H5 HPAI (A), H5 HPAI 2.3.2.1 clade (B), and H5 HPAI 2.3.4.4 clade (C) in field-infected feces samples. The validity of this assay was confirmed using M-gene positive field samples, H5Nx HPAI viruses for positive control, and Newcastle Disease virus for negative control. The results were visualized using an intuitive heat-map (green color-positive result, red color-negative result). Each concentration had triple replicates. (D) Clade classification of HPAI H5 subtype viruses from field-infected feces samples. The HA1 partial sequences of the HA gene with multiple basic cleavage sites were obtained by conventional RT-PCR and all H5 HPAI-positive sequences were verified using the Influenza Research Database (www.fludb.org).

The one-step multiplex RT-qPCR assay for the detection of pan-H5 HPAI (A), H5 HPAI 2.3.2.1 clade (B), and H5 HPAI 2.3.4.4 clade (C) in field-infected feces samples. The validity of this assay was confirmed using M-gene positive field samples, H5Nx HPAI viruses for positive control, and Newcastle Disease virus for negative control. The results were visualized using an intuitive heat-map (green color-positive result, red color-negative result). Each concentration had triple replicates. (D) Clade classification of HPAI H5 subtype viruses from field-infected feces samples. The HA1 partial sequences of the HA gene with multiple basic cleavage sites were obtained by conventional RT-PCR and all H5 HPAI-positive sequences were verified using the Influenza Research Database (www.fludb.org).