Establishment of a quantitative real-time PCR assay for the quantification of apple proliferation phytoplasmas in plants and insects.

摘要

A quantitative PCR (qPCR) assay was established for a sensitive and specific quantification of apple proliferation (AP) phytoplasmas in plants and insect vectors (Cacopsylla picta). Different AP phytoplasma-specific primer pairs previously selected in a non-ribosomal DNA fragment of AP phytoplasma were tested. Among these, primer pair AP3/AP4 was chosen for the qPCR assay because it amplifies a small sized 162 bp fragment of AP phytoplasma and produces no artefact bands. Thus, with these primers, the SYBR Green technology could be used to monitor the amplification of the PCR products in real-time. The absolute quantification of the phytoplasmas in the samples was done by using the standard curve quantification method. The plasmid pUCI196 containing the chromosomal fragment of AP phytoplasmas from which the specific primers were derived was used as standard. Serial dilutions of the plasmid were done in total DNA extracts of healthy plants and healthy psyllids, respectively. For insects, total DNA of single individuals was extracted and subjected to PCR. Thus, AP phytoplasmas could be quantified in single individuals. For plant material, quantification of AP phytoplasmas was done with reference to a defined fresh weight of the material prior to DNA extraction. The inter-assay and intra-assay reproducibility of the method was analysed by comparing the Ct-values for given samples. The reproducibility was high both with plant and insect samples. Great differences in phytoplasma load could be found in different insect vector individuals whereas the analysed plant material was more homogenously infected. The established method is now suitable for the study of the infectivity of the insect vectors as well as for the evaluation of the resistance in plant material.