Development and Application of a Real-Time Reverse-Transcription PCR and Droplet Digital PCR Assays for the Direct Detection of Potato mop top virus in Soil

Potato mop top virus (PMTV) is a continuing threat to potato production throughout the world. It has the potential to persist in the soil for long periods in the sporosori of its vector Spongospora subterranea f. sp. subterranea, which is as an important source for PMTV infection and dissemination. In this study, we used real-time quantitative reverse-transcription PCR (qRT-PCR) and reverse-transcription droplet digital PCR (RT-ddPCR) assays of the total RNA extracted directly from the soil to develop a simple, fast, and sensitive method to detect PMTV in soil samples using a specific primer with high efficiency despite a minimal amount of viral RNA. The designed primers are resilient in the presence of various PCR inhibitors in the soil when RNA is extracted. Both assays detected PMTV in all soil types used and supported the detection of <10 PMTV copies mu l(-1) in the RNA sample. With qRT-PCR, detection was linear, with amplification efficiencies ranging from 93.3 to 105.3% for silt loam, loamy sand, sand, and sandy loam in various experiments with R-2 > 0.99. Furthermore, the RT-ddPCR assay also demonstrated a high degree of linearity (R-2 > 0.99 and P < 0.0001) with the RNA extracted from the soil samples representing different textures and physiochemical characteristics that were artificially spiked with infested S. subterranea f. sp. subterranea sporosori. Additionally, both assays successfully detected PMTV in different types of naturally infested soil with PMTV carrying S. subterranea f. sp. subterranea sporosori levels ranging from 6.2 x 10(2) g(-1) to 1.2 x 10(6) g(-1) in soils with pH ranging from 4.9 to 7.5 and organic matter ranging from 0.9 to 5.1%, demonstrating the potential to detect PMTV in a wide variety of soils. To our knowledge, this is the first report of the development of real-time PCR and ddPCR methods for the direct detection of a soilborne virus in soil.