Establishment And Application Of The RT-RPA And Fluorescent RT-PCR Method For Detection Of Avian Influenza Virus

Avian Influenza(AI)is a severe infectious disease syndrome caused by avian influenza virus belonging to the genus Influenza A of Orthomyxoviridae,which seriously endangers animal health and human health.Influenza A virus is the most harmful andmutant influenza virus,which can evade the host immune response,so it is difficult to control.In 1878,it was reported for the first time that bird flu broke out in chicken flocks.It still affects the health of poultry on a global scale,causing huge economic losses to the poultry industry and threatening human life.In recent years,the diagnostic technology of avian influenza has developed rapidly,especially in the field.Recombinant Enzyme Polymerase Amplification(RPA)has the advantages of simple operation method and fast reaction speed,and the amplification of nucleic acid can be completed in 20 minutes at 37?42?.By selecting the M gene sequence of influenza A virus,the HA gene sequence of H5 subtype and H7 subtype of avian influenza virus and the HA and NA gene sequence of H7N9 subtype of avian influenza virus,we designed many pairs of primers and probes,and successfully established the RT-exo RPA of avian influenza virus.The detection method was compared with the fluorescent PCR method.The test results show that the method is lower sensitive than the real-time RT-PCR,but it is faster,more sensitive and easier to operate.In 2013,a new strain of avian influenza H7N9,which can infect humans,broked out in Yangtze river delta region.Since the outbreak of H7N9 avian influenza,more and more cases of human deaths caused by avian influenza have caused great harm to the development of poultry industry and social stability.In 2017,the death toll from H7N9 avian influenza in China rose sharply,causing another global sensation.In this stduy,a dual fluorescence RT-PCR method for detection of highly pathogenic H7 subtype avian influenza virus was successfully established.The sensitivity and specificity of the method were analyzed through three batches of tests of the dual fluorescence RT-PCR detection for H7 subtype avian influenza virus.The test results show that the detection sensitivity of this method was10^1.869EID₅₀ for highly pathogenic avian influenza virus(H7 subtype)and 10^1.940EID₅₀ for low pathogenic avian influenza virus(H7 subtype).The method can specifically distinguish between H7 subtype avian influenza virus and highly pathogenic H7 subtype avian influenza virus,with strong specificity.This method can be used for epidemiological investigation of H7 subtype avian influenza.