Premium
A Simple and Fast Extraction Procedure to Obtain Amplifyable DNA from Ralstonia (Pseudomonas) Solanacearum and Clavibacter michiganensis ssp. sepedonicus Inoculated Potato Tuber Extracts and Naturally Infected Tubers to Conduct a Polymerase Chain Reaction (PCR)
Author(s) -
Niepold F.
Publication year - 1999
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1046/j.1439-0434.1999.147004249.x
Subject(s) - clavibacter michiganensis , ralstonia solanacearum , biology , bacterial wilt , dna extraction , bacteria , extraction (chemistry) , inoculation , microbiology and biotechnology , potato starch , food science , starch , polymerase chain reaction , horticulture , pathogen , chromatography , biochemistry , gene , chemistry , genetics
A modified NaOH alkaline boiling procedure using a mixture of lysozyme and proteases combined with minimized TRIS/HCl/BSA buffer volume was applied to extract amplifyable DNA from the two quarantine bacteria Ralstonia (Pseudomonas) solanacearum and Clavibacter michiganensis ssp. sepedonicus artificially added to potato tuber extracts of a low and a high starch potato variety. A PCR detection threshold of 10 4 −10 5 colony forming units per ml extract of each quarantine bacterium was obtained by using the two potato varieties, the high starch potato variety resulting in a lower detection threshold. Similar sensitivities could be obtained from potato tubers naturally infected with both quarantine bacteria. When comparing a published DNA extraction procedure suitable for Clavibacter michiganensis ssp. sepedonicus with the alkaline extraction the latter is much faster and simpler with similar detection thresholds and representing an inexpensive method to obtain suitable template DNA for routine PCR tests.