Diagnosis of Sugarcane White Leaf Disease Using the Highly Sensitive DNA Based Voltammetric Electrochemical Determination

摘要

A simple and highly sensitive analysis by electrochemical voltammetry has been developed for diagnosis of the most destructive crop disease in Thailand known as sugarcane white leaf (SCWL). Determination of the corresponding DNA interaction has been obtained from the voltammetric signals of electroactive redox methylene blue (MB) by means of cyclic and differential pulse voltammetry. In this study, a chitosan-modified glassy carbon electrode (GCE) was created by self-assembly to produce electrostatic platform for effective immobilization of the DNA. Fabrication of SCWL-DNA hybridization detection system was performed by immobilizing the ssDNA probe as a specific sensor onto chitosan-modified GCE. Hybridization of complementary DNA from the real samples could then be detected by its respective MB signal. This fabricated DNA probe sensor was shown to be capable for discriminative identification among the DNAs from SCWL plants, mosaic virus infected sugarcane and healthy sugarcane plants. Relationship between the specific hybridization signal and DNA target concentration was also observed under optimal condition. The detection limit of 4.709 ng/μl with the regression coefficient (R2) of 0.998 and overall RSD of 2.44% were obtained by response curve fit analysis. The actual SCWL-ssDNA immobilization and hybridizing event were subsequently confirmed by an observation under atomic force microscope. Thus these experiments demonstrate the first successful and effective DNA based voltammetric electrochemical determination for a verification of the specific pathogenic infection within plants from the real epidemic field.