Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by Multiplex PCR with Coamplification of Host DNA

Abstract

A new multiplex PCR assay was developed for the detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers. The assay combines two different tests in one reaction mixture. First, a highly specific and sensitive detection of the pathogen and second, an indicator test for successful amplification (internal PCR control), which monitors potentially false-negative PCR results, caused by inhibition of the PCR. For the simultaneous amplification of two different targets in one reaction mixture, a mix of two different primer sets was used. For the detection of C. michiganensis subsp. sepedonicus, a pathogen-specific primer set PSA-1/PSA-R was used, based on the intergenic spacer region of the 16S–23S rRNA genes of C. michiganensis subsp. sepedonicus. For the simultaneous amplification of the internal PCR control, the plant-specific primer set NS-7-F/NS-8-R was employed, permitting amplification of target sequence from plant DNA present in DNA extractions from potato core fluid. The applicability of the multiplex PCR was verified in 3500 composite samples of 200 seed potato tubers from 143 different cultivars in a survey for C. michiganensis subsp. sepedonicus by parallel testing using immunofluorescence, a bioassay in eggplant seedlings and multiplex PCR.