Reseach On Immunosorbant Assay And Gene-chip Methods For Seedborn Bacterias In Corn Seeds

Bacterial Wilt of Corn or Stewart's Wilt (Pantoea stwartii), and Goss's bacterial wilt and blight of maize (Clavibacter michiganensis subsp. nebraskensis, Cmn ) are important disease classes in corn production. Currently they are taking places in most part of North America.Therefore these two bacterial diseases have already recoreded in been in quarantine test list for Chinese entry plant, despite of that they have not been still reported in China. Nowadays, it is very important to establish a sensitive and efficient testing method for the inspection and detection of the couple of corn diseases.There are three detection methods for these two bacterium which are separated culture, serological and PCR detection method.There are no reports of the Cmn monoclonal antibodies in serological detection. Compared with the polyclonal antibodies, monoclonal antibodies showed specificity, high purity and stability. Microarray method is one of the world's most advanced core technology of portable biochemical analyzer, Comparing with routine separated culture and PCR detection methord, Gene-chips expressed a high detection sensitivity, high throughput and high parallelism capacity, which can meet with a precise detection requirement. Therefore, in this article, on basis of Cmn monoclonal antibodies, we established a double-monoclonal-antibody sandwich ELISA method for detection of the Cmn, which filled up the blank of monoclonal-antibody detection methods in-around the world, and settled the basis for preparation of ELISA kit and test strip in the next step. By Gene-chip study on the two quarantine bacterium of maize and related bacteria, a Geng-chip detection method was developed, the method can realize simultaneous detection of multiple bacterium.The results were summarized as follows:1 Subclasses, activity, and specificity of the Cmn-McAb were test, and the optimum condition for antigen and antibody were screened.2 A noval DAS-ELISA method was established with its minimum detection limit of the bacteria was 1.0×108 CFU/L, and the bacteria in the seed suspension with high repeatability and stability had been detected.3 Primers and probes for Pantoea stwartii, Cmn, Clavibacter michiganensis subsp.michiganensis (Cmm) and Clavibacter michiganensis subsp.insidiosus (Cmi) were designed. And a specific and sensitive multiple PCR of the bacteria was developed.4 The visual gene chips were preparation and the best conditions for the detection were screened.5 A specific and sensitive visual Gene-chips detection methord was established. It can simultaneous of two or more individual bacteria. Little influences in matrix during the process of sample testing were observed.In this study, we developed two new detection methods of DAS-ELISA and Gene-chip for corn bacteria diseases. They can meet with part of the requirements for entry maize testing. The detection sensitivity and efficiency of gene chip are better than the ELISA method. Yet the ELISA method is relatively cheap. In order to reduce testing cost, ELISA method can be used firstly with the redetection for the positive samples arranging in pair with the two detection methods to achieve accurate, efficient and economic detection results.