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

. CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/566281 doi: bioRxiv preprint michiganensis (Fig 4; Table 3 ). Although the overall mechanism involved in improved sensitivity and efficiency due to the use of flap primers in TaqMan qPCR assays is still not completely clear, a study conducted by Vandenbussche and group (2016) [31] suggests that tailed primers suppress the formation of PCR artifacts, determined by primers and dNTP type. Also, tailed primer reactions respond differently to the use of hot-start dNTPs. In this study, the use of the TaqMan real-time qPCR master mix containing hot-start dNTP led to specific detection and amplification without the report of either PCR artifacts or shifting in the sigmoidal shape of the amplification curves.