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

In this study, we conducted detailed in vitro and in silico analyses designed to enhance the effect of tailed primers in TaqMan real-time qPCR assays that simultaneously detect all nine subspecies of C. michiganensis as well as specifically identifying C. m. subsp. nebraskensis. (Table 1) . Strain designations beginning with "A" were obtained from the Pacific Bacterial Collection at the University of Hawaii at Manoa. Bacterial cultures were taken from -80°C; gram-positive bacteria were grown on TZC-S medium (peptone 5 g/L, 10 g/L, sucrose 5 g/L and agar at 17 g/L, 0.001% 2,3,5-triphenyl-tetrazolium chloride) and gram-negative bacteria were grown on TZC medium with 5 g/L dextrose as the carbon source and 0.001% tetrazolium chloride) [19] -plates were incubated at 26°C (±2°C). Grown cultures were used for DNA isolation using Wizard Genomic DNA Purification Kit following the manufacturer's instructional manual (Promega, Madison, WI).