Rapid detection of Candidatus Phytoplasma mali by recombinase polymerase amplification assays

Isothermal recombinase polymerase amplification (RPA) assays for the specific detection of Candidatus Phytoplasma mali (Ca. P. mali), the causal agent of apple proliferation, were developed. The assays amplify a fragment of the imp gene and amplimers were detected either by fluorescence in real-time mode (TwistAmp (R) exo assay) using a fluorophore-labelled probe or by direct visualization employing a lateral flow device (TwistAmp (R) nfo assay/Milenia (R) HybriDetect). The RPA assays specifically amplified DNA from Ca. P. mali strains, and cross-reactivity with other phytoplasmas or plant DNA was not observed. The limit of detection was determined with a cloned imp standard, and positive results were obtained down to 10 copies with both RPA assay formats. In comparison with a TaqMan real-time PCR assay based on the same target gene, the RPA assays were equally sensitive, but results were obtained faster. Simplified nucleic acid extraction procedures from plant tissue with Tris-and CTAB-based buffers revealed that crude Tris-DNA extracts were a suitable source for RPA tests while larger concentrations of CTAB were inhibitory. This is the first report of RPA-based assays for the detection of Ca. P. mali. The assays are suitable for high-throughput screening of plant material and point-of-care diagnostic and can be potentially combined with a simplified DNA extraction procedure.