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

To determine sensitivity and PCR yields, multiplex end-point PCR and TaqMan real-time qPCR assays were performed employing two sets of primers with and without 5' AT-rich sequences ( Table 2 ). Sensitivities were determined using 10-fold serially diluted genomic DNA; the DNA concentrations ranged from 10 ng to 10 fg per reaction. Pure bacterial DNA from C. m. subsp. nebraskensis (A6206) was used for all sensitivity assays; NanoDrop v.2000 spectrophotometer (Thermo Fisher Scientific, Inc., Worcester, MA) was used to measure DNA concentrations. All TaqMan qPCR reactions were performed in three replicates. The spiked assays were performed by adding 1 µl of healthy corn host DNA into TaqMan qPCR sensitivity reactions prepared using serially diluted (10 ng to 10 fg) genomic DNA of C. m. subsp. nebraskensis (A6206).