Rapid detection of Cucumber mosaic virus isolates representing distinct phylogenetic subgroups by reverse transcription, loop-mediated isothermal amplification

Several outbreaks of Cucumber mosaic virus in cucurbit crops have been observed recently in Poland. Based on the phylogenetic analysis of the genes' encoding movement and coat protein, Polish CMV isolates from zucchini were classified into two subgroups: IA and II. A reverse transcription, loop-mediated amplification method (RT-LAMP) was developed for rapid and effective detection of genetically diverse CMV isolates. RT-LAMP was performed with a set of six primers, the design of which was based on the coat protein gene. Positive effects of the RT-LAMP were visualized by direct staining of the reaction with fluorescent dyes, agarose gel electrophoresis, and analysis of the amplification curves in real-time conditions. The RT-LAMP method developed here was capable of the detection of diverse CMV isolates in less than 1 h. The sensitivity of RT-LAMP was tenfold higher than that of conventional RT-PCR.