Development of High Resolution DNA Melting Analysis for Simultaneous Detection of Potato Mop-Top Virus and Its Vector, Spongospora subterranea, in Soil.

In this study, a set of duplex reverse transcription (RT)-PCR-mediated high resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f.sp. subterranea (Sss), was developed. The infestation of soil by PMTV was detected by using a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel-electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, -CP, and -TGB) were analyzed together with the existing Sss primers using real-time RT-PCR. The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by high resolution DNA melting (HRM) analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV and/or Sss was found in 63-100% samples collected from fields in which PMTV-infected tubers had been observed. In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Between 63%-83% and 100% of the soil samples collected from PMTV-infested fields led to PMTV and Sss infections in the bait tobacco plants, respectively; whereas no PMTV or Sss infected plants were obtained from soil samples collected from PMTV/Sss-free fields.