Visual isothermal amplification for rapid detection and genotyping of avian influenza virus

Objective To establish a rapid nuclear-acid detection of the avian influenza virus and three subtypes including H5N1,H7N7 and H9N2.Methods A conserved region of the M gene in all avian influenza viruses was amplified to detect the avian influenza virus.The subtypes of avian influenza virus were further identified by amplifying a subtype-specific sequence of HA gene.The assay was performed by a highly sensitive loop-mediated isothermal amplification(LAMP) incubated at 60 ℃ for 1 hour with three pairs of LAMP primers and Bst DNA polymerase.The visual detection was readily realized through the incorporation of SYBR Green I fluorescence to the LAMP reaction system.Results The sensitivity of detecting three subtypes was 10 DNA copies per tube,and there was no non-specific amplification among the detection of the three virus subtypes.Both of the accuracy and the specificity were 100% in 18 negative samples and 2 positive samples.Conclusion The established rapid detection is suitable for point-of-care tests of avian influenza virus and three subtypes.