ISHS Acta Horticulturae 825: I Balkan Symposium on Fruit Growing
COMPARISON OF METHODS OF ISOLATING DNA FOR ESFY PHYTOPLASMA DETECTION |
| Authors: | V. Masková, B. Krska, T. Necas |
| Keywords: | phytoplasma ESFY, DNA extraction, non-specific primers, nested PCR, apricot |
| Abstract:
‘Candidatus phytoplasma prunorum’, (European stone fruit yellows phytoplasma - ESFY), is nowadays one of the most important diseases belonging to the list of organisms for which quarantine is required, and is a long-standing problem in apricot and peach orchards in the Czech Republic. Its detection and diagnosis is based on molecular methods, especially "nested PCR". There are various methods for the isolation of DNA from leaf-stalks and the phloem (from stalk or roots), and four different methods were tested: Lee et al. (1991), two commercial DNA isolation kits (Dneasy® Plant Maxi Kit and NucleoSpin® Plant XL) and, as a comparison, the method described by Ahrens and Seemüller (1992). For the nested PCR detection of ESFY we used non-specific primers like R16F1/R16R0 (out-primers) and fU5/rU3 (in-primers). The results demonstrate that none of the methods used for the DNA isolation and subsequent PCR reactions provide the consistent quality and quantity of DNA which is necessary for ESFY phytoplasma detection. | |
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