Using sigmoidal curve-fitting in a real- time PCR detection assay to determine detection thresholds

Abstract

Phytophthora ramorum, the causal agent of sudden oak death (SOD) is a quarantine pathogen that has forced the implementation of extraordinary measures to track and contain the movement of infected nursery stock both within and outside of the three western states of California, Oregon and Washington. Federal guidelines in the United States for diagnostic testing of P. ramorum are in place to insure the sensitivity and reliability of detection tests. PCR assays are used to determine whether the Phytophthora sp. detected by the initial immunoassay screening is P. ramorum. Most of the time definitive results can be obtained from a single PCR reaction. However, there are times when the accuracy of the results can be called into question because of low pathogen titer, a situation that can result in false negatives given the limits of detection of the marker system. In addition, experimental samples often contain significant amounts of PCR inhibitors that can also give results outside the normal detection cutoffs. Therefore the need for re-testing the samples using the same or alternative methods of pathogen detection is manifest. To understand the effect of low DNA concentration, or the role of PCR inhibitors in the extracted DNA in the sensitivity of detection of P .ramorum, plant and pathogen specific markers were amplified in real time PCR experiments using TaqMan® chemistry. The kinetics of amplification f the PCR reactions were modeled using a four-parametric sigmoidal curve. Standard curves of pure P. ramorum DNA and plant host DNA, with standard amounts of P. ramorum DNA added, were created and used to establish a base for data analysis. Samples having low pathogen titer were amplified and the values of the sigmoid curve parameters described. Statistical analysis of data allowed the identification of samples falling outside the proposed 95 percent confidence interval. Curve modeling also provided experimental support for determining threshold values for assessing the presence of the pathogen.

Parent Publication

• Proceedings of the sudden oak death third science symposium

Citation

Uribe, Pedro; Martin, Frank N. 2008. Using sigmoidal curve-fitting in a real- time PCR detection assay to determine detection thresholds. In: Frankel, Susan J.; Kliejunas, John T.; Palmieri, Katharine M., tech. coords. 2008. Proceedings of the sudden oak death third science symposium. Gen. Tech. Rep. PSW-GTR-214. Albany, CA: U.S. Department of Agriculture, Forest Service, Pacific Southwest Research Station. pp. 75-81