Development and Application of Droplet Digital PCR Assay for the Detection of Watermelon Silver Mottle Virus and Melon Yellow Spot Virus

Abstract

Watermelon silver mottle virus (WSMoV) and melon yellow spot virus (MYSV) (Tospoviridae, Orthotospovirus) are responsible for silver mottle mosaic and yellow spot symptoms, posing threats to melon (Cucumis melo), watermelon (Citrullus lanatus), and cucumber and leading to significant economic losses in China. Early disease detection and monitoring of these two viruses are necessary for disease management, for which a rapid, reliable, and adaptable diagnostic method is required. In this study, using a droplet digital PCR (ddPCR) method, the conserved region of the nucleocapsid gene (N gene) sequence was detected in WSMoV and MYSV. The probes and primers for WSMoV and MYSV did not detect other relevant cucurbit viruses, and the specificity reached 100%. Although both qPCR and ddPCR exhibited good reproducibility, the reproducibility of ddPCR was better than that of qPCR. The reproducibility of ddPCR was proved to be 100%. Moreover, ddPCR exhibited a good linear correlation with varying concentrations of targets. The detection limits of WSMoV and MYSV in ddPCR were 18 and 9 copies/μL and were approximately 12- and 18-times more than those in qPCR, respectively. Finally, 62 samples collected from the field (including infected melon, watermelon, and weeds) were further evaluated for the presence of WSMoV and MYSV. The field samples exhibited 91.94% and 51.61% positivity rates in ddPCR assays for WSMoV and MYSV, respectively; the rates were higher than those in qPCR (59.68% and 43.39%, respectively). The results indicated that ddPCR has a higher accuracy than qPCR. Therefore, ddPCR could be used in the clinical diagnosis of early infections of WSMoV and MYSV. To the best of our knowledge, this is the first study to establish a ddPCR method for the detection of WSMoV and MYSV. The application of this method for differential detection of MYSV and WSMoV will help in understanding the epidemics caused by these two important viruses and provide important information for the early detection, monitoring, and rapid extermination of infection.