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

Each primer and probe set ( Table 2 ) was designed to be used in both endpoint PCR (endpoint PCR products between 100 to 200 bp) and TaqMan real-time qPCR (both multiplex and single reaction). Broad range detection capabilities and specificity of primers and probes were tested against inclusivity and exclusivity panels containing twenty-nine strains of C. michiganensis subspecies and twenty-five gram-positive and gram-negative bacteria from either symptomatic corn leaf or closely related plant associated bacteria (S1 Table) . Fourteen strains and two naturally infected samples were unmistakably detected as C. michiganensis subsp. nebraskensis using primer Cmn11-F/R and probe Cmn11-P; no cross-reactivity was observed with any other non-target species/subspecies (Table 1) . Likewise, the primer (CM-F/R) and probe (CM-P) set exhibited an accurate and specific detection of all twenty-nine strains corresponding to the nine subspecies of C. michiganensis; no cross-reactivity with the members of exclusivity panel was detected. As expected, 151-and 107-bp PCR amplicons were amplified in multiplex endpoint PCR for C. michiganensis subsp. nebraskensis and Clavibacter michiganensis species, respectively (S3 Fig). All primers and probes met the desired 100% query coverage and 100% identity after an alignment using BLASTn against the NCBI nucleotide and genome databases (Table 2) . Thermodynamics, internal structures, GC% content and Δ G values for each primer and probe were calculated using mFold and Primer3 Plus; all the parameters are listed in Table 2 . The tendency of primers to form self-structures increased when additional nucleotides were added to the primers; nevertheless, no interference in primer annealing with the target sequence was observed when 5' AT-rich sequences were added to the primers. Furthermore, assay sensitivity for primer sets with flaps was increased ten-fold suggesting positive impact on reaction thermodynamics and stability during annealing and extension steps (Fig 2) .