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Enhanced Polymerase Chain Reaction Methods for Detecting and Quantifying Phytophthora infestans in Plants

October 2000 , Volume 90 , Number  10
Pages  1,112 - 1,119

Howard S. Judelson and Paul W. Tooley

First author: Department of Plant Pathology, University of California, Riverside 92521; and second author: U.S. Department of Agriculture, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, 1301 Ditto Ave., Ft. Detrick, MD 21702

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Accepted for publication 26 June 2000.

Sensitive and specific primer sets for polymerase chain reaction (PCR) for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any system.

The American Phytopathological Society, 2000