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

Population genetic and comparative genomic analyses are imperative components for the development of robust and specific diagnostic tools (5, 26, 27, 28) . Multiple genome alignments using Mauve software resulted in highly conserved and unique gene regions specific for C. michiganensis in general (major facilitator superfamily gene) and specifically for C. m. subsp. nebraskensis (core sugar ABC transporter permease and ABC transporter ATP-binding genes). Besides the selection of unique and conserved targets, primer and probe design plays a very important role in developing a robust and specific multiplex TaqMan qPCR assay [17, 29] . Assay specificity can also be enhanced using dual-quenched probes [16, 30] ; that is, an internal quencher, TAO or ZEN, was added to each probe to reduce the distance from reporter to quencher, leading to low background signal and greater precision ( Table 2 ).