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

Arif and Ochoa-Corona (2013) [17] found that the addition of non-complementary AT-rich sequences at the 5' position of the forward and reverse primers enhanced the detection sensitivity and overall PCR amplicon yields; they reported that the addition of customized sequences on suboptimal (thermodynamically incompetent) primers increased the PCR yield by about 20% and sensitivity by 10-100 folds. In the current study, we observed that the addition of 5' AT-rich sequences not only increased the sensitivity but also optimized the reaction efficiency from 82% to 99% and 91% to 100% for C. michiganensis in general and C. michiganensis subsp. nebraskensis, respectively (Fig 4) . One hypothesis is that addition of 5' AT-rich sequences to the primers enhanced the reaction thermodynamics allowing for faster primer annealing to the target template resulting in optimal reaction efficiency; however, the exact mechanism is unknown. The outcome of flap addition also resulted in a 10-fold increase in sensitivity for detection of C.