| Clavibacter michiganensis subsp.Nebraskense and Pantoea stewartii subsp.Stewartii are the two plant pathogen, which can cause tremendous agricultural economic losses.The methods for the detection of the two pathogen have isolation culture detection, ELISA and PCR. However, these methods are time-consuming, require skilled technicians and special equipment. It's difficult to apply rapidly in spot. Immunochromatographic assay which is simple, rapid and easy to analyze has been widely used for rapid detection in pathogen, but the assay is limited sensitivity.Facing the shortage of the immunochromatographic assay in detection of Clavibacter michiganensis subsp.Nebraskense and Pantoea stewartii subsp.Stewartii, a technology system was drawn up using nano-particles colloidal gold enhancement method. This study was developed the method for detection of the two pathogen.1.The preparation conditions of 40nm colloidal gold were successfully established by a modified sodium citrate reduction method. Studies were explorated on the optimal conditions about the colloidal gold labeled with monoclonal antibodies, such as monoclonal antibody 4G12 against Clavibacter michiganensis subsp.Nebraskense, monoclonal antibody 2F5 against Pantoea stewartii subsp.stewartii. After the the establishment of the colloidal gold conjugates with the monoclonal antibodies conditions (the optimum pH is 8.0～8.5, the optimum amount of monoclonal antibody is 30μg/mL and 15μg/mL respectively),colloidal gold-4G12 conjugate and colloidal gold-4G12 conjugate were prepared.2.The colloidal gold immunochromatographic strip was studied for the detection of Clavibacter michiganensis subsp. Nebraskense. The assay was based on sandwich format using two monoclonal antibodies,4G12 labeled by gold nanoparticles that was served as a detection reagent,4H4 immobilized in a defined detection zone on a nitrocellulose membrane. The positive standard strain was detected by the prepared strip, yielding a strong red color line, while the control was negative. The sensitivity was 106cfu/mL, after the enhancement,105cfu/mL of Clavibacter michiganensis subsp.Nebraskense was detected in short time. The immunochromatographic strip was 10 times sensitive as the double antibodies sandwich ELISA. Meanwhile, no cross-reaction with other 9 strains was found, indicating good specificity of the strip,and showing good repeatability and stability. Results were shown that the strip can be applied in rapid detection with high sensitivity and specificity.3.The colloidal gold immunochromatographic strip was studied for the detection of Pantoea stewartii subsp.Stewartii. The assay was based on sandwich method using two antibodies,the monoclonal antibody 2F5 labeled by gold nanoparticles that was served as a detection reagent, the polyclonal antibody immobilized in a test line on a nitrocellulose membrane. The sensitivity was 106cfu/mL, after the enhancement,104cfu/mL was detected, indicating the sensitivity was improved 100 fold. Meanwhile, no cross-reaction with other 9 strains was found, showing good specificity of Pantoea stewartii subsp. Stewartii strip. As a rapid and convenient method for the detection Pantoea stewartii subsp.Stewartii, the development of immunochromatographic strip has practical significance for agricultural and food safety.
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