| Sumario: | Loop-mediated Isothermal Amplification (LAMP) assays for detection of potato strain (sequevar 1) (LAMP I), Ralstonia solanacearum species complex (RSSC) strains (LAMP II) and Ralstonia solanacearum (Rs) phylotype I (LAMP III) have recently been developed. A study was conducted to validate real-time fluorescence detection of RSSC strains from soil, potato stem and tuber extracts using alkaline PEG extraction method employing LAMP I, LAMP II and LAMP III assays in laboratory for routine diagnostics. The stem and tuber samples were collected from farmers’ fields in Elgeyo Marakwet county in Kenya. The soil sample was obtained from Kenya Plant Health Inspectorate Service and it was inoculated with pure Rs cultures grown on CPG media before extracting the crude DNA using alkaline PEG. LAMP I was specific to Sequevar 1 and 2 RSSC strains, LAMP II detected phylotype I, II, and III tested strains whereas LAMP III detected phylotype I strains as expected. The sensitivity of the assays was determined to be at 103 CFUs/ml for stem, tuber and soil samples for pure cultures. However, the LOD was further reduced to 10 CFUs/ml and the time to positivity (Tp) reduced to 5 mins for crude DNA extracted using alkaline PEG after spiking the different CFUs into potato tuber. This is the first report of RSSC strains detection from soil samples using LAMP assay. Samples that tested positive for Rs bacteria using LAMP were confirmed to be positive with NCM ELISA. The simplicity of sampling process, running the assay, time to result of under an hour, use of handheld field deployable device makes the assay an attractive alternative to standard ELISA test for seed certification under resource limited context.
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