A simple and visible detection method for the rapid diagnosis of Ustilaginoidea virens in rice seeds by a loop‐mediated isothermal amplification assay

Rice false smut is an important rice seed-borne disease caused by the fungus Ustilaginoidea virens. It is vital to develop assays for the detection and identification of U. virens in rice seeds. Here, we present a simple and visual detection method for the rapid diagnosis of U. virens in rice seeds based on a loop-mediated isothermal amplification (LAMP) assay. This LAMP assay was optimized using a set of six primers targeting a specific region of the APN1 (aminopeptidase N1) gene, which encodes a peptide-terminated enzyme. Compared with the previously reported LAMP methods, a metal ion indicator dye hydroxy naphthol blue (HNB) was added to the prereaction solution in this study to reduce the contamination risks. Among the genomic DNA of 5 strains of U. virens and 13 other fungi, only the five tubes containing U. virens DNA showed a colour change from violet to sky blue. The sensitivity of the LAMP assay with the HNB dye was 100 pg of DNA, which was 10 times more sensitive than that of conventional PCR. This LAMP approach was also verified by testing rice seed samples, including infested seeds, and the U. virens in the rice seeds could be detected. These results indicate that this LAMP assay with a group of six primers targeting a specific region of the APN1 gene is a simple and visual detection method that can be applied for the rapid detection of U. virens in rice seeds.