POSTERS: Pathogen detection, quantification and diagnosis
Development and application of a real-time RT-PCR assay for the direct detection of potato mop top virus in soil
Binod Pandey - Department of Plant Pathology, North Dakota State University. Ipsita Mallik- Department of Plant Pathology, North Dakota State University, Neil Gudmestad- Department of Plant Pathology, North Dakota State University
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 (Sss) which serve as an important source for PMTV infection and dissemination. In this study, we used real-time reverse transcription PCR of the total RNA directly extracted from the soil to develop a simple, fast and sensitive method to detect PMTV. The effort was made to identify a sensitive and specific primer with high efficiency despite a minimal amount of viral RNA or the possible presence of various PCR inhibitors in soil extracted RNA. The assay detected PMTV from all soil types used and it supported the detection of less than 10 PMTV copies µl-1 in the RNA sample. Detection was linear with amplification efficiencies ranging from 93% to 105% for silt loam, loamy sand, sand and sandy loam in various experiments with R2 values >0.99. Additionally, the assay developed successfully detected PMTV from different types of naturally infested soil with PMTV carrying Sss sporosori ranging from 6.2 × 102 g-1 to 1.2× 106 g-1 in soils with pH ranging from 4.9 to 7.5, and organic matter ranging from 0.9% to 5.1% indicating this assay has the potential to detect PMTV from a wide variety of soil.