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

Accurate detection and identification are critical in diagnosis and microbial forensics for disease control and management. The use of a robust multigene format maximizes broad-range detection capabilities, reliability and specificity for C. michiganensis species detection. Fifty-nine strains representing different diseases and hosts were included in the inclusivity and exclusivity panelsno false positives or false negatives were detected. Of 59 samples, 29 were identified as members of various C. michiganensis subspecies, 16 were identified as C. m. subsp. nebraskensis and 25 samples (other than Cm and Cmn) showed no reactivity when specific TaqMan qPCR assays were performed ( Table 2 ). The positive qPCR results with genomic DNA of twenty-nine diverse strains in the inclusivity/exclusivity panels collected from different geographical locations across USA and the world confirmed specificity and broad-range identification of C. michiganensis subspecies and C. m. subsp. nebraskensis using the novel primer and probe sets developed in the present study (Table 1) .