Synergetic effect of non-complementary 5’ AT-rich sequences on the development of a multiplex TaqMan real-time PCR for specific and robust detection of Clavibacter michiganensis and C. michiganensis subsp. nebraskensis

Adriana Larrea-Sarmiento; Anne M. Alvarez; James P. Stack; Mohammad Arif

The developed TaqMan qPCR assays have proven to be truly sensitive and efficient for simultaneous and single detection of C. michiganensis and C. m. subsp. nebraskensis. The assays were fully validated with well-described strains in inclusivity and exclusivity panels. The universal primer set P16-F/R designed to amplify the 16S rRNA partial gene region of plantassociated bacteria provided genus and species identification for the eleven bacterial pathogens from corn, including gram-positive and gram-negative bacteria (S1 Table) . Furthermore, primer set CM-dnaA-F/R, designed to amplify the dnaA partial-gene sequence of C. michiganensis, was used to confirm the identity of all nine subspecies (S1 Table) . The housekeeping dnaA gene has been proven as a potential genetic marker for plant bacterial phylogenetic studies and identity confirmation [5] .