Development of Rapid Assay for Avian Influenza A(H5N1) Virus by Loop-Mediated Isothermal Amplification Method

Objective:To set up loop-mediated isothermal amplification(LAMP) method for quick detection of H5N1 avian influenza A virus.Methods:The hemagglutinin gene of avian influenza virus subtype H5 was downloaded from GenBank and analyzed using MegAlign tools of software DNAStar.The outer, inner and loop primers of LAMP were then designed according to their conservative region using software PrimerExplorer V3.The fragment of subtype H5 sequence was also amplified and cloned to serve as the positive standard and optimized its LAMP reaction condition and reaction system in this experiment.Results:The results showed that the LAMP assay had a detection limit of 4～6 DNA copies.The primers used in this assay showed the specificity for H5N1 avian influenza A virus and did not amplify H1 virus or H9 or New Castle disease virus.Conclusion:It is proved that LAMP method is a reliable and accurate assay for quick detection of H5N1 virus.This assay offers a new method and idea for detecting avian influenza virus.