Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi

Abstract

Real-time PCR technologies open increasing opportunities to detect and study phytopathogenic and antagonistic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing real-time analysis of the reaction kinetics and allowing quantification of specific DNA targets. Four main chemistries are currently used for the application of this technique in plant pathology. These chemistries can be grouped into amplicon sequence non-specific (SYBR Green I) and sequence specific (TaqMan, Molecular beacons, and Scorpion-PCR) methods. Amplicon sequence nonspecific methods are based on the use of a dye that emits fluorescent light when intercalated into doublestranded DNA. Amplicon sequence specific methods are based on the use of oligonucleotide probes labelled with a donor fluorophore and an acceptor dye (quencher). The fluorescent signal eliminates the requirement for post-amplification processing steps, such as gel electrophoresis and ethidium bromide staining. This significantly reduces time and labour required for the analysis and greatly increases the throughput of PCR testing as an automated diagnostic system, making it suitable for large-scale analyses. Furthermore, the use of different fluorescent dyes facilitates the detection of several target microrganisms in a single reaction (multiplex-PCR). Real-time PCR makes possible an accurate, reliable and high throughput quantification of target fungal DNA in various environmental samples, including hosts tissues, soil, water and air, thus opening new research opportunities for the study of diagnosis, inoculum threshold levels, epidemiology and host–pathogen interactions. Moreover, the quantification of specific mRNA transcription by real-time PCR is being increasingly applied to the study of changes in gene expression in response to phytopathogenic and antagonistic fungi.