Quantitative analysis of HA gene of human influenza H1N1 virus by PCR method

Influenza disease is one of the fatal diseases in human and birds,that several pandemic of it has lead to high mortality in all over the world.Nuraminidase Antigene (NA) and Hemaglutinine Antigene (HA) are couple of the most important surface antigenes of the virus ,because they involve recombination and changes during the virus evolution.These alterations has cause several epidemic and pandemic of the influenza virus to take place until.one way to evaluate the rate of the influenza virus is quantitative analysis of the influenza virus HA gene by Real time PCR.In this study quantitative analysis of H1N1 human influenza virus HA gene was optimized by Real time PCR. PCR was performed using primers designed for HA gene fragment. The HA gene fragment was inserted into the PTG-19 vector and the cloning was confirmed by sequencing assay and digestion by BamH1 enzyme.The plasmid containing HA gene was diluted in one-tenth dilutions and quantitative PCR based on SYBR green was performed for them. The plasmid containing HA gene was created and confirmed by sequencing of HA gene and enzymatic digestion assays and evaluated by Real time PCR based on SYBR green in different dilutions and the difference of CT between two dilutions was about 3.5.Sensitivity of the optimized assay was 170 copy number of DNA in each reaction. By optimizing of quantitative analysis of HA oncogene by Real time PCR based on SYBR green,were obtained conditions that make it possible to evaluate the expression of HA gene in clinical sampels and so in cell cultures treated by candidate drugs of Influenza virus.

Keywords:

H1N1 human influenza virus , HA surface antigene , quantitative Real time PCR