Adaptation of real-time PCR assay for specific detection of apple proliferation phytoplasma.

摘要

A real-time PCR assay was developed for the specific detection of apple proliferation (AP) phytoplasma. A previously designed MGB probe (qAP-16S) was evaluated on our phytoplasmas collection (mainly AP, PD [pear decline] and ESFY [European stone fruit yellows]). Using this probe, late fluorescent curves were obtained from ESFY isolates. These curves crossed the threshold calculated as 10 x SD. Thus, a sequence alignment was made using database sequences and sequencing results. A new MGB probe was designed in a region presenting a single nucleotide polymorphism between AP and ESFY isolates. Using this probe, a specific detection of AP was obtained irrespective of the method of threshold calculation. No late amplification was observed with other phytoplasmas. The amplification of serial dilutions of initial template DNA showed that this method is at least 16- and 8-fold more sensitive than conventional PCR with specific (AP5/AP4) and polyvalent (qAP-16S-F/R) primers, respectively. Phytoplasma infection on inoculated apple trees was more rapidly detected using this new probe in real-time PCR than by conventional PCR or biological indexing. The optimization of an existing method resulted in a rapid, specific and sensitive method for the detection of AP.