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TECHNICAL SESSION: Advanced detection and diagnosis of plant diseases

One lab-one protocol: synergetic effect of 5’AT-rich flap to harmonize qPCR protocols for easy, sensitive and cost-effective diagnostics
Mohammad Arif - University of Hawaii at Manoa.

Accurate and rapid diagnostics streamline efficient disease management and can minimize losses due to diseases. In diagnostic labs, hundreds of protocols are in place to detect different pathogens from different hosts. In this research, our aim was to devise a single qPCR protocol that can be used for different pathogens. Customized non-complementary AT-rich flap sequences were added at the 5’ position of each primer to improve the reaction thermodynamics, and then developed TaqMan qPCR protocols that worked under the same condition for Clavibacter michiganensis, C. michiganensis ssp. nebraskensis, C. michiganensis subsp. michiganensis, Dickeya, D. dianthicola, D. solani, Pectobacterium, P. parmentieri and a universal internal control (UIC). The primer and probe sets for these pathogens can be mixed and matched based on the user’s preference without the need for further validation with exclusivity and inclusivity panels. Also, the inclusion of 5’ AT-rich sequences enhanced the qPCR reaction efficiency from 0.82 (M = -3.83) and 0.91 (M = -3.54) to 1.04 (optimum slope value; M = -3.23) for both C. michiganensis and C. michiganensis ssp. nebraskensis, respectively; an increase of 10-fold sensitivity was also obtained with C. michiganensis. No false positives or negatives were detected. The proposed methodology will improve existing protocols and reduce the labor, processing time and reagent consumption; overall the diagnostic cost per sample will be reduced.