Development of a duplex TaqMan real-time PCR for the general detection of phytoplasmas and 18S rRNA host genes in fruit trees and other plants

Abstract

The detection of phytoplasmas in fruit trees and other important crops is demanded from nurseries and certification agencies. We have therefore developed an efficient assay for general detection of phytoplasmas. This duplex assay combines the phytoplasma detection with an internal control, detecting host gene 18S rRNA in the same reaction. The internal control enables the confirmation of an efficient DNA extraction and the recognition of eventual inhibition of the PCR. In contrast to a cytochrome oxidase (COX) assay, which did not work in fruit trees, 18S rRNA host gene detection was shown to be usable in a broader range of plants. The sensitivity and robustness of the duplex qPCR was evaluated with different dilutions of samples and compared with traditional nested PCR. The duplex qPCR was at least as sensitive as nested PCR and less susceptible to inhibition due to impurities in DNA extracts. The method was successfully employed for the screening of phytoplasmas in samples of fruit tree, raspberry, and grapevine. Typically, phytoplasma positive samples were tested with Ct values of 22-30, whereas Ct values for the internal control were in the range of 13-23.