Research Of The Inspection Method For Clavibacter Michiganensis Subsp. Nebraskensi

The below determination methods of Clavibacter michiganensis subsp. Nebraskense were developed as Molecular Biology detection,Serological detection, isolation and culture detection. However, the disadvantages of these methods such as time-consuming, skilled technicians and special equipments required were the main reasons which made them difficult to be applied in rapidly determination.In this research, a series of novel methods in determinating Clavibacter michiganensis subsp. Nebraskense were developed. The results of the methods are listed as below:1. An indirect Enzyme-Linked Immunosorbnent Assay method for determination of Clavibacter michiganensis subsp. Nebraskense has been developed. By using this method, the limit of detection was1×106CFU/mL.2. Double antibody sandwich biotin-avidin ELISA (BAS-ELISA) procedure was developed for Clavibacter michiganensis subsp. Nebraskense determination. The limit of detection of this method was1×105CFU/mL which was an order of magnitude higher than indirect ELISA.3. The preparation conditions of40nm colloidal gold were successfully established. The method involved modified microwave synthesis. The optimization of the conditions about the colloidal gold labeled with monoclonal antibodies, such as monoclonal antibody4H4against Clavibacter michiganensis subsp. Nebraskense has been processed. The conditions of colloidal gold conjugated with the monoclonal antibodies were optimized,(the optimum pH was8.0～8.5, the optimum amount of monoclonal antibody was24μg/mL)and colloidal gold-4H4conjugate were prepared.4. 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 in which4H4labeled by gold nanoparticles was prepared as detection reagent and4G12immobilized in a defined detection zone on a nitrocellulose membrane was as capture antibody. The limit of detection for Clavibacter michiganensis subsp. Nebraskense was1×106CFU/mL.