Rapid detection of "Candidatus Phytoplasma mali" by recombinase polymerase amplification assay

Valasevich, Natalia; Schneider, Bernd GND

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®exo assay) using a fluorophore-labelled probe or by direct visualization employing a lateral flow device (TwistAmp®nfo ssay/Milenia®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.

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Valasevich, Natalia / Schneider, Bernd: Rapid detection of "Candidatus Phytoplasma mali" by recombinase polymerase amplification assay. 2017.

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