Studies On Detection Of Pseudomonas Syringae Pv. Tomato In Tomato Seed Using Immunomagnetic Separation And The Polymerase Chain Reaction

Tomato bacterial speck caused by Pseudomonas syringae pv. tomato (PST) is an important disease for the production and the quality of tomato. It has become a problem in the north of China recent years. One important aspect of the disease is that the pathogen is naturally borne and transmitted by seed which usually serves as the primery inoculum source for TBS. So it is an important task to find out a strategy which requires rapid, reliable, and sensitive seed-detection assays.The study was carried on at key laboratory of the nation at IPP CAAS. Two antigen was obtained through two method that implemented in this study and three kind of serum was obtained according to the two ways of injection. The reaction of antigen with antibody was carried out in tube: the antibody of rabit anti whole cell of PST could also be effective when diluted by 640 times; the other two kinds of antibody can be effective after diluted by 1280 times. The speciality of serum acquired by ELISA showed that the speciality of all 3 serum are almost the same. The best one is rabit anti PST18, which has the fewest false positive reaction. The next one is the serum of rabit anti whole cell of PST. The reaction between the serum of rabit-anti-PST2844-87 and the nonPST has the largest number.The DNA of 74 strains of bacteria in this study were extracted via the method of CTAB DNA extraction. Followed by the CTAB DNA extraction PCR was employed. Two pairs of specific primers were synthesized based on the published sequence of some specific gene in PST. And the parameter of PCR cycle was determinated subsequently. The pair of the primer(3) and primer(4) is more specific than the pair of primer(1) and primer(2). Application of IMS as a diagnostic tool in clinical microbiology veterinary medicine and food microbiology has been studied extensively. In this study we have done some basic work to prepare for the using of IMS in detection of seedborn PST.We've obtained the magnetic microsphere whose nucleus is Fe3O4 with Agar next to it, which serves as steady spherical surface. The beads were keeped at 4 . We've concentrated the antibody to prepare for the coating of the magnetic microsphere.In the detection of PST in tomato seed using ELISA and PCR, we've employed a series of percentage of polluted seeds. The threshold of polluted seed recovery by ELISA is 1% and by PCR is 0.1%.