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.
|Titolo:||Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi|
SCHENA, Leonardo (Corresponding)
|Data di pubblicazione:||2004|
|Appare nelle tipologie:||1.1 Articolo in rivista|