Barley yellow mosaic disease, caused mainly by barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV), is a devastating disease of barley and is a threat to Eurasian barley production. Early detection is essential for effective management of the pathogens and to assure food security. In the present study, a simple, rapid, specific, sensitive, and visual method was developed to detect BaYMV using loop-mediated isothermal amplification (LAMP). Two pairs of oligonucleotide primers (inner and outer primers) were designed to amplify the gene encoding the coat protein of BaYMV. The optimal conditions for the LAMP method were determined, and a one-step reverse transcription (RT)-LAMP method was also developed. Subsequently, the fastest processing time for RT-LAMP was determined. Among eight plant viruses examined using the LAMP method, only BaYMV was detectable, suggesting that the assay was highly specific. The RT-LAMP method was ten times more sensitive than the RT-PCR method in the sensitivity test. To further shorten the virus detection process, a dye was added to the RT-LAMP products, and positive reactions were simply read by the naked eye via a color change (from orange to light green) under visible light. Barley samples from the middle and lower reaches of the Yangtze River basin, where barley yellow mosaic disease broke out very seriously in 1970s, were detected by the newly established RT-LAMP method. The results showed that all samples were positive for BaYMV, indicating that the potential risk of the virus in these areas. This newly established LAMP/RT-LAMP method could be a promising tool for barley protection and food security control.
Development of reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) for the detection of Barley yellow mosaic virus
