Loop-mediated isothermal amplification assay: A specific and sensitive tool for the detection of Bipolaris oryzae causing brown spot disease in rice

Rice brown spot caused by Bipolaris oryzae (Breda de Haan) is the predominant disease in rice, infecting millions of hectares of cultivated rice, and greatly reduces yields. The various available diagnostic methods for B. oryzae disease detection are time consuming and require sophisticated instruments. Thus, an affordable, rapid and highly specific assay is needed. Here, we report a loop-mediated isothermal amplification (LAMP) assay protocol for specifically detecting B. oryzae by targeting a gene encoding a glycoside hydrolase (GH) family 13 protein. A set of six specific, sensitive LAMP primers were designed, allowing the effective LAMP detection of pathogens after incubation at 62°C for 60 min. Visual detection was performed by the addition of hydroxynapthol blue dye. In a positive reaction, the color changed to intense sky blue, while in a negative reaction, the color remained violet. The developed LAMP assay was highly sensitive, with a detection limit of 100 femtograms. The high specificity of the assay for the detection of B. oryzae was validated. Thus, the LAMP assay protocol provides a rapid, reliable tool for the early detection of B. oryzae under laboratory conditions.