One-Step Multiplex Quantitative RT-PCR for the Simultaneous Detection of Viroids and Phytoplasmas.

A one-step multiplex quantitative reverse transcription polymerase chain reaction protocol is described, for the detection in pome trees of Pear blister canker viroid and Apple scar skin viroid, together with universal detection of phytoplasmas. Total nucleic acids extraction is performed according to a modified CTAB protocol and TaqMan MGB probes are used to surpass high genetic variability of viroids. The multiplex real-time assay is at least ten times more sensitive than conventional protocols and its features make it suitable for rapid and massive screening of pome fruit trees phytoplasmas and viroids in certification schemes and surveys.