Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes

In recent years quantitative PCR (qPCR) detection methods have been widely utilised to detect phytopathogenic fungi and oomycetes and have greatly contributed to the advancement of knowledge in the plant pathology sector. However, major drawbacks and common errors, most typical of earlier reports, still affect many methods currently available in literature. Errors can be made during the entire complex process for the development of qPCR methods which includes the selection of appropriate DNA extraction and purification protocols, the identification of suitable target regions, the choice of the chemistry, the design and validation of specific primers and probes, the analysis of sensitivity, the choice of an absolute and/or relative quantification approach and the analysis of the risk of detecting target DNA from dead material. In the present review above mentioned phases are accurately analysed in order to highlight critical aspects and provide a practical guide for final users.