Detection of mRNA by reverse transcription PCR as an indicator of viability in Phytophthora ramorum

Abstract

Real-Time PCR technologies offer increasing opportunities to detect and study phytopathogenic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing both real-time analysis of the reaction kinetics and quantification of specific DNA targets. Before the development of Real-Time PCR and the opportunity to provide quantitative data, the risk of false-positive PCR results due to detection of dead cells was considered only a minor setback. This, therefore, has led to a renewed interest in the risk of false-positive PCR results. In order to deal with this potential problem we developed a new reverse transcript (RT) PCR assay based on the use of mRNA as a viability marker, on the basis of its rapid degradation compared to DNA. We developed new primers, specific for P. ramorum, designed in the cytochrome oxidase gene encoding subunits I (COXI). To evaluate the specificity of the method, four isolates of P. ramorum and 11 different Phytophthora species were tested. One hundred symptomatic bay leaves from three different sites in California were collected in three different seasons of the year. Samples were plated on PARP selective media for Phytophthora and tested with the new RT-PCR method and compared with a TaqMan and SybrGreen Real-Time PCR assay after DNA extraction. Results showed that after seven days RNA of freeze-dried killed P. ramorum was undetectable while DNA gave a positive signal. Furthermore, data from the new assay were more correlated to the results obtained after isolation on selective media whereas DNA-based results showed more positive samples. This indicates that by using the new RT-PCR method, the risk of false-positive PCR results due to detection of dead cells can be minimized.

Parent Publication

• Proceedings of the sudden oak death third science symposium

Citation

Chimento, Antonio; Cacciola, Santa Olga; Garbelotto, Matteo. 2008. Detection of mRNA by reverse transcription PCR as an indicator of viability in Phytophthora ramorum. In: Frankel, Susan J.; Kliejunas, John T.; Palmieri, Katharine M., tech. coords. 2008. Proceedings of the sudden oak death third science symposium. Gen. Tech. Rep. PSW-GTR-214. Albany, CA: U.S. Department of Agriculture, Forest Service, Pacific Southwest Research Station. pp. 85-92