Enhanced polymerase chain reaction methods for detecting and quantifying Phytophthora infestans in plants

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 perfor mance of these primers was compared to those developed previously for the i nternal transcribed spacer (ITS) of ribosomal DNA. The detection limit usin g the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is abo ut 100 times more sensitive than ITS-directed primers. Nested polymerase ch ain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using th e new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic als o served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimensio n of this study was that two laboratories independently checked the specifi city and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any syste m.