Influenza Viral Genome Sequencing

The single-infection isolates were further confirmed by plaque-purificationas previously described^{43 (link)}, and then used for subsequent sequencing. Mixed-infection isolations were verified by real-time RT-PCR as previously described^{44 (link)}. Viral RNA was extracted from viral stock fluid using an EasyPure Viral DNA/RNA kit (TransGen, Beijing, China) according to the manufacturer’s manual. cDNA was synthesized from viral RNA by reverse transcription with the 12-bp primer 5'-AGCAAAAGCAGG-3' as previously described^{45 (link)}. PCR was performed using specific primers as described in previous research to obtain the full-length HA and NA genes^{46 (link)}. The PCR products were purified with the TaKaRa Agarose Gel DNA Purification Kit Ver. 2.0 (TaKaRa, Dalian, China) and sequenced by Invitrogen of Guangdong Co., Ltd. A BLAST search was performed to compare the sequences against the nucleotide sequences of all known HA and NA genes of AIV in the GenBank database, and the HA and NA subtypes of the isolates were determined and verified.

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Luo S., Xie Z., Li M., Li D., Xie L., Huang J., Zhang M., Zeng T., Wang S., Fan Q., Zhang Y., Xie Z., Deng X, & Liu J. (2021). Survey of low pathogenic avian influenza viruses in live poultry markets in Guangxi Province, Southern China, 2016–2019. Scientific Reports, 11, 23223.

Publication 2021

EasyPure Viral DNA/RNA kit TaKaRa Agarose Gel DNA Purification Kit Ver. 2.0

Agarose Cdna Genes Infection Isolations Mixed infection Nucleotide sequences Plaque Primers Real time pcr Reverse transcription Viral dna Viral rna

Corresponding Organization :

Other organizations : Guangxi Veterinary Research Institute

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Variable analysis

independent variables

Viral RNA extraction using an EasyPure Viral DNA/RNA kit
CDNA synthesis from viral RNA by reverse transcription with the 12-bp primer 5'-AGCAAAAGCAGG-3'
PCR amplification using specific primers to obtain the full-length HA and NA genes

dependent variables

Sequencing of the PCR products to determine the HA and NA subtypes of the isolates

control variables

Plaque-purification of single-infection isolates as previously described in the referenced study
Verification of mixed-infection isolations by real-time RT-PCR as previously described in the referenced study

controls

Positive control: Previously described plaque-purification method for single-infection isolates
Positive control: Previously described real-time RT-PCR method for verification of mixed-infection isolations

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