Improved real-time PCR assay for detection of the quarantine potato pathogen, Synchytrium endobioticum, in zonal centrifuge extracts from soil and in plants |
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Authors: | Marga P. E. van Gent-Pelzer Marjon Krijger Peter J. M. Bonants |
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Affiliation: | (1) Plant Research International BV, P.O. Box 69, 6700 AB Wageningen, The Netherlands; |
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Abstract: | Real-time PCR was used for quantitative detection of the potato pathogen, Synchytrium endobioticum, in different substrates: zonal centrifuge extracts, warts and different plant parts of potato. Specific primers and a TaqMan probe, designed from the internal transcribed spacer region of the multi-copy rDNA gene were tested in extracts from artificially and naturally infested soil. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable by guarding against false negative results. A calibrations curve was created by spiking zonal centrifuge fractions of clean soil samples with a dilution series of winter spores. The Taqman assay was also performed on infected potato plant material (stolons) along with the detection of the cytochrome oxidase gene as a potato endogenous control. Sensitivity of the TaqMan assay was improved at least 100-fold and proved to be reliable for accurate diagnosis of the disease. |
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