Barcoding Detection Technology Research In Influenza A Virus

In the recent years, DNA barcoding technology is one of the most rapidly developing skills which can identification of species or types rapidly and accurately by applying a standard DNA fragment. DNA barcoding applying for influenza study of discriminating different subtypes is a new concept, which is still mainly used in identifying animal species. Due to the nucleotide mutation of influenza A virus, finding a suitable DNA barcoding is very difficult. In this study, three groups of DNA barcoding (HA cleavage sites, HAO, HA2 and NAb) we are found not only can amplified effectively respectively for subtype identification distinction, also acquire some genetic information. The purpose of this study was to finding an suitable barcoding sequence which both conservative enough to design universal primers and sufficient variation to distinguish different subtypes between closely related subtypes,the results were as follows:1. We established optimality genome amplification conditions of A\swine\Shandong\01\2009(H1N1)、 A/equine/Heilongjiang/01/2010(H3N8)、A/swine/Shandong/2/03(H5N1)、A/chicken/Shandong/sx01/2008 (H9N2) influenza virus strains, and analysisd their own sequence characteristics, combined with Sanger sequencing.2. Choosing hemagglutinin cleavage site as the target area, we found an barcoding primer that can distinguish 16 different subtypes.By RT-PCR, an approximately 170bp fragment was obtained. Afterwards we constructed the phylogenetic tree and calculated the average intraspecific variation and interspecific divergence by MEGA5.0 software. Phylogenetic trees presented that amino acid composition of cleavage site has a characteristics of monophyly in each antigenic subtype. The average interspecific divergence is 0.277, intraspecific variation is 0.032. It is conclused that DNA barcoding technology can be used effectively in the molecular identification of the influenza A virus subtypes.3. By bioinformatics analysising of 16 hemagglutinin (HA) and 9 neuraminidase (NA), degenerate primers were designed according to the conserved region of HA2 and NAb as the barcode primer, and then barcoding library preparation was optimized, In the end ,we acquiced reads covered all 25 subtypes of by NGS.And it provides a simple diagnosis and high-throughput sequencing system.