Reverse transcription loop-mediated isothermal amplification assay for the rapid and simultaneous detection of H5 and other subtypes of avian influenza viruses

A two-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was designed for the rapid visual detection of the M gene of all subtypes of avian influenza virus (AIV) and the H5 gene of the H5 subtype of highly pathogenic AIV (HPAIV). The reaction carried out in two tubes in a single step at $58^{\circ}C$ for 40 min, and the assay results could be visually detected by using hydroxynaphthol blue dye. Using M or H5 gene-specific primers, the assay successfully detected all subtypes or H5 subtypes of AIVs, including the Korean representative H5N1 and H5N8 HPAIVs. The detection limit of the assay was approximately $10^{2.0}$ $EID_{50}/reaction$ for the M and H5 genes of H5N1 HPAIV, respectively, and was more sensitive than that of previously reported RT-LAMP and comparable to that of real-time RT-PCR. These results suggest that the present RT-LAMP assay, with its high specificity, sensitivity, and simplicity, will be a useful diagnostic tool for surveillance of currently circulating H5 HPAIVs and other subtypes of AIV in bird population, even in under-equipped laboratories.