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Comparison of Dig-Labeled PCR, Nested PCR, and ELISA for the Detection of Clavibacter michiganensis subsp. sepedonicus in Field-Grown Potatoes

March 2001 , Volume 85 , Number  3
Pages  261 - 266

I.-M. Lee , L. A. Lukaesko , Molecular Plant Pathology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705 ; and C. J. M. Maroon , Agdia Inc., 30380 County Road 6, Elkhart, IN 46514

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Accepted for publication 20 October 2000.

The sensitivity of digoxigenin- (dig-) labeled polymerase chain reaction (PCR) was compared with nested PCR and enzyme-linked immunosorbent assay (ELISA) for the detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus, in seed potatoes and stem tissues. The bacterial DNA was extracted from chopped tuber or stem tissue by a modified hot alkaline DNA extraction method. C. michiganensis subsp. sepedonicus specific DNA sequence was amplified by dig-labeled PCR with the primer pair CMSIF1/CMSIR1 previously designed based on the insertion element IS1121 of C. michiganensis subsp. sepedonicus. Dig-labeled PCR products were then blotted on a nylon membrane and the signal was detected by a colorimetric assay using alkaline phosphatase. The new assay procedure has a detection sensitivity close to that of nested PCR and simplicity of the standard ELISA procedure commonly used commercially. The dig-labeled PCR assay was more sensitive than ELISA and can be used to detect C. michiganensis subsp. sepedonicus in symptomless field potato tubers.

Additional keywords: Clavibacter michiganensis subsp. insidiosus, threshold values

The American Phytopathological Society, 2001