A new method to develop internal controls for the determination of phytoplasma concentration

Mergenthaler, E. and Fodor, M. and Süle, S. (2004) A new method to develop internal controls for the determination of phytoplasma concentration. Acta Phytopathologica et Entomologica Hungarica, 39 (1-3). pp. 1-7. ISSN 0238-1249

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Abstract

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.

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