Real-Time PCR Detection Methods for Economically Important Grapevine Related Bacteria

Abstract

Preventive measures are extremely important for control of diseases that are caused by plant pathogenic bacteria on economically important plants. For this reason, fast and reliable detection methods are required. Along with the time-consuming conventional detection methods such as isolation and culturing of bacteria on media, PCR-based methods have been introduced as supplementary tests for better diagnosis of plant pathogenic bacteria. In the case of non-culturable bacteria, such as Phytoplasmas, PCR-based tests even became indispensable. Real-time PCR has become a widely used platform in nucleic acid detection and quantification in diagnostics. Despite the general guidelines for some of the steps, there are no common guidelines or standard operating procedures for introduction of real-time PCR-based detection systems for pathogens for routine laboratory use. Four cases of detection systems for grapevine pathogenic bacteria that were developed at the National Institute of Biology are presented and discussed here, providing a practical overview of the whole process from the initial assay design to the implementation of the assay into the laboratory.

Primers designed for the detection of grapevine pathogens spreading with propagating material by quantitative real-time PCR

“…The quantitative real-time PCR: Soon after the introduction of conventional PCR a more developed technique, the quantitative real time-PCR (q-PCR) has also been introduced for the detection of plant pathogens (Boonham et al 2014, Christensen et al 2013, Hren et al 2010, Schena et al 2004. The q-PCR is more sensitive than the conventional PCR method and the amplification and detection is combined in one step, thus subsequent gel-electrophoretic separation of the amplified product is not necessary.…”

Bacterial diseases of grapevine

“…Several conventional and real-time PCR-based protocols have been described for the detection and identification of these bacterial pathogens (Hren et al 2010, Palacio-Bielsa et al 2009b. The application of multiplex PCR allows the identification of genetically diverse types of bacteria in a single reaction (Bini et al 2008, Kawaguchi et al 2005, Pulawska et al 2006, while the use nested or semi-nested PCR (Peduto et al 2010, Pulawska & Sobiczewski 2005 increases the specificity and sensitivity of detection.…”