A one-step reverse-transcription loop-mediated isothermal amplification assay optimized for the direct detection of cucumber green mottle mosaic virus in cucurbit seeds

The Molecular Evolutionary Genetics Analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

Cucumber green mottle mosaic virus (CGMMV) was first described in 1935 infecting cucumber, making it one of the first plant viruses to be studied. Its initial distribution occurred out of England to other countries. This was followed by its distribution from England and these other countries to additional countries. This process increased slowly between 1935 and 1985, faster between 1986 and 2006, and rapidly between 2007 and 2016. The discovery that it diminished cucurbit fruit yields and quality, especially of watermelon, prompted a substantial research effort in worst-affected countries. These efforts included obtaining insight into its particle and genome characteristics, evolution, and epidemiology. CGMMV's particle stability, ease of contact transmission, and seed transmissibility, which are typical tobamovirus characteristics, explained its complex disease cycle and its ability to spread locally or over long distances without a vector. Knowledge of its disease etiology and epidemiology enabled development of integrated disease management approaches that rely heavily on diverse phytosanitary measures. Dispersal of seed-borne infection through the international seed trade following cucurbit seed crop production in tropical or subtropical countries explains its recent rapid dispersion worldwide.