This study aimed to develop a quick and simple method that generates internal controls from any polymerase chain reaction (PCR) amplified phytoplasma DNA fragment. Non-specific PCR conditions were used to generate unspecific DNA bands with the same primers as the target phytoplasma DNA fragment, but different in their sizes. The method is universal enough to be adaptable for any selected primer pairs. The procedure does not require preliminary knowledge of the sequence or restriction sites of the amplified DNA fragment. Developed internal controls are ligated into a bacterial vector, which can serve as a competitor, to co-amplify with the target phytoplasma DNA in a competitive PCR reaction. Serial dilutions of the internal controls with adjusted concentration and fixed amounts of target templates from phytoplasma-infected plants were amplified together with the same primers to estimate the relative amount of phytoplasma DNA.
The tuf gene of “Candidatus Phytoplasma mali”, the causal agent of apple proliferation was PCR cloned in an expression vector and expressed in Escherichia coli. First, phytoplasma DNA extracted from periwinkle was amplified using primers designed on the basis of the tuf gene and the PCR product was cloned into pGEM-T (Promega). In the next step specific primers were constructed containing some plasmid sequences and restriction enzyme sites. With this primers the sequence in pGEM-T was amplified, the product was digested with restriction enzymes, and inserted into the pQE40 expression vector (Qiagen). In this plasmid the tuf gene was fused to the 6xHIS tag, and DHFR. The production of 6xHIS-DHFR-Tu fusion protein protein was induced with IPTG and expressed in E. coli M15. The new fusion protein was found in the insoluble fraction of the bacterium. The identity of the protein was verified with polyacrylamid gel-electrophoresis and Western blot analysis using antiserum raised against the 6xHIStag of the fusion protein.