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

Real-Time qPCR is a powerful and robust technique and offers greater sensitivity and speed compared with conventional PCR; however, qPCR sensitivity can be compromised as a result of poor primer thermodynamics. Primer thermodynamics can be improved by adding 5' AT-rich sequences often called overhang, flap, or tail [16, 17, 18] ). In addition, primer Tm and length can also be fine-tuned to harmonize the amplification reaction conditions [16, 17] . The tailed primers have been reported to provide higher PCR yields and sensitivity [16, 17, 18] . Unfortunately, the underlying mechanism behind the action of flap primers is still unknown.