A comparison of different phytoplasma DNA extraction methods using competitive PCR

Abstract

The primer pair 16endF-Tmod, specific for the16S/23S rDNA spacer region, amplifies different length fragments when the target DNA belongs to phytoplasmas of the X-clade and AP-clade in compared with phytoplasmas of the AY-clade. The cloned 16S/23S spacer of the tagete witches’-broom phytoplasma (TWB, AY-clade) was used in a competitive PCR assay to quantify the DNA of the apple proliferation phytoplasma (AP, AP-clade) and the clover phyllody phytoplasma (CP, X-clade) present in the nucleic acids extracted from infected periwinkle plants. Thus, four methods, normally used for the extraction of phytoplasma DNA from infected plant tissue, were compared to determine which was the most efficient in recovering phytoplasma nucleic acids. The different methods detected relatively minor differences in the yields obtained. It was concluded that the sampling and detection protocols are more critical than the DNA extraction method as far as sensitivity is concerned.