Real-time polymerase chain reaction: applications to studies on soilborne pathogens

Real-time, or quantitative, polymerase chain reaction (Q-PCR), offers a rapid, sensitive, and specific method for the diagnosis of plant pathogens in soil, water, air, and plant samples. Fluorescence is used to monitor the accumulation of the PCR product after each PCR cycle. The fluorescence data are used to extrapolate the amount of target DNA present in the sample before amplification so that detection and quantification are achieved in a single assay. Detection limits are generally in the femtogram range for purified pathogen DNA, in the picogram range for pathogen DNA in plant samples, and one to several spores for soil samples. Quantification is reliable over a dynamic range of five to seven orders of magnitude of target DNA, in which differences of two fold or more in target DNA can be detected. Real-time PCR has already been recognized as a valuable tool for epidemiological studies, disease management, detection of emerging pathogens, evaluation of resistance and tolerance to disease, and phytosanitary screens. In this review, we compare four major real-time chemistries that have been used for the detection and quantification of soilborne plant pathogens and discuss the advantages and limitations of TaqMan (TM) and SYBR (TM) green 1, the most widely used chemistries. Current applications of real-time PCR to the diagnosis of plant pathogens in soil and plant samples, considerations for assay development, and variations in measurements are also discussed.