Development of High-Resolution DNA Melting Analysis for Simultaneous Detection of Potato Mop-Top Virus and Its Vector, Spongospora subterranea, in Soil
| Autor: | Vikram Bisht, Cassandra Gilchrist, Mathuresh Singh, Yasmine Soqrat, Jacques Lavoie, Manisha Shukla, Dahu Chen, Bihua Nie, Virginia Dickison, Xianzhou Nie, Alexa Creelman |
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| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine Gel electrophoresis Veterinary medicine biology Total rna High resolution Spongospora subterranea Plant Science Amplicon biology.organism_classification 01 natural sciences SSS 03 medical and health sciences Potato mop-top virus Nucleic acid thermodynamics 030104 developmental biology Agronomy and Crop Science 010606 plant biology & botany |
| Zdroj: | Plant Disease. 105:948-957 |
| ISSN: | 1943-7692 0191-2917 |
| Popis: | In this study, a set of duplex reverse transcription PCR (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 with 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, RNA-CP, and RNA-TGB) were analyzed together with the existing Sss primers via 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 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 or Sss was found in 63 to 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. Of the soil samples collected from PMTV-infested fields, 63 to 83% and 100% 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- and Sss-free fields. |
| Databáze: | OpenAIRE |
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