Establishment Of A Fluorescence Quantitative PCR Method For Detecting Phylogenetic Lineages Of H1N1 Influenza Virus

H1N1 influenza virus is an important member of influenza A virus family,and mammals are more susceptible to it than other Influenza subtypes in mammals.H1N1influenza virus is also the main subtype causing pandemic and seasonal human influenza.In recent years,the probability of isolation of H1N1 virus from poultry,especially in wild and live poultry markets,has been increasing.The public threat and economic loss posed by the subtype of H1N1 virus have far exceeded other subtypes of influenza.At present,H1N1 influenza virus has established a relatively stable phylogenetic lineagein human,mammalian and poultry populations.Therefore,understanding the infection and epidemic situation of phylogenetic lineage ofH1N1influenza virus will be of great benefit to us in identifying the lineage source of influenza virus,identifying the dominant lineage that is prevalent in each population,selecting vaccines matching the antigenicity of influenza lineage and screening vaccine viruses.In this study,the phylogenetic analysis of 2037 HA genes of H1N1 subtype influenza virus isolated from China in GenBank and gisaid database revealed that the virus has evolved four different phylogenetic lineages.namely,classical swine influenza virus lineage,seasonal human influenza virus lineage,avian influenza virus lineage and Eurasian avian-like swine influenza viruses lineage.Based on the phylogenetic analysis results of the evolutionary tree,we compared the nucleotide sequences of HA genes in each lineage,selected the relatively conservative region of HA gene,designed multiple pairs of primers for each lineage,and proposed to establish a fluorescent quantitative PCR detection method for the phylogenetic lineageof H1N1 influenza virus.Meanwhile,the positive standard plasmids of each lineagewere constructed as the positive standard of this method.The establishment process of this method is as follows:Using the positive standard plasmids of each lineage as the template,the identification primer of each lineage was initially screened by common PCR method,and then the reaction system and reaction conditions were optimized.The screened primers of each lineage were respectively used for quantitative PCR experiments.By constructing a standard curve,the identification primers of each lineage were re-screened,and the reaction system and conditions optimized by common PCR methods were verified.The experimental results showed that when the reaction system of fluorescence quantitative PCR was:FastStart Universal SYBR Green Master?ROX?10?L,ddH₂O8.75?L,0.5?L of the upstream and downstream primers at a concentration of 10?M,0.25?L of five standard samples with different copy numbers(10¹⁰copies/?L?10⁸copies/?L?10⁶copies/?L?10⁴copies/?L?10²copies/?L),to reach the final volume of 20?L;the reaction conditions were:the first stage,95?for 2min,the second stage,40 cycles of 95?for 15s,47?for 30s.During 40 cycles,the melting curve generated by the screened identification primers of each lineage was a single peak type,and there were no non-specific miscellaneous peaks;and the Ct values detected using standard plasmids with different copy numbers in each lineage were evenly distributed on the standard curve,showing a good linear relationship?both R²?0.945?.The detection limit of each lineage identification quantitative PCR method was 10²copies/?L,showing that the established method has good sensitivity.On this basis,laboratory preserved and clinical isolated subtypes of influenza virus were used as the test object to explore the specificity of the established method.The identification primers of each lineage screened in this study can only bind to its corresponding lineage H1N1 subtype influenza virus template,and there is no cross-reaction with other lineage H1N1 subtype influenza viruses or H3N2,H9N2 and other subtype influenza virus templates,indicating the established method has better specificity.Therefore,the fluorescence quantitative PCR method established in this study for the phylogenetic lineage identification of H1N1 influenza viruses has a relatively ideal sensitivity and specificity,and can quickly identify the lineage of H1N1influenza viruses.