Studies On Detection Techniques Of Serology & PCR And The Genetric Diversity Of Clavibacter Michiganensis Subsp. Michiganensis

The serology,PCR methord,the genetric diversity of Clavibacter michiganensis subsp.michiganensis (abbreviation Cmm)had been studied.The main results are as follows:To use the protein of Cmm immuned rabbit and gained the antiserum.The titer of antiserum is 1:80 after agar double diffusion method detecting.After ammonium sulphate precipitation,salting out,the purification of sephadex gel laminar analysis,the antiserum can specificly detect Cmm,and can classify Clavibacter michiganense subsp.Sepedonicum and Clavibacter michiganense subsp.Insidiosum. Compared with indirect ELISA and the Cmm ELISA kit made in Agdia company,proved that the specificity of the indirect ELISA is higher ELISA kit,consuming time is shorte,and have the same sensitivity.The indirect ELISA can be used in the fiel and lab teseting.To assess the adaptability and efficiency of traditionary PCR and real-time fluorescent PCR(TaqMan) for detection of Clavibacter michiganensis subsp.michiganensis in bacteria suspension from pure culture and the extracts DNA from naturally infested or infectted tissues,a comparative study was conducted.The results showed that the threshold of traditionary PCR testing is 10⁵cfu/mL,the threshold of treal-time fluorescent PCR(TaqMan)is 10^3-4cfu/xnL.Compared to two PCR testing methords,the real-time fluorescent PCR(TaqMan)didn't requrie agarose gel electrophoresis,ethidium bromide staining and Southern blotting.And it had much higher sensitivity,accouting for 10-100 times,but requried expensive instruments and reagent,and it was adapted to lab testing and basal research.Rep-PCR analysis for thrity-three isolates of Cmm collected from several proveinces and cities in the whole country was conducted.The results showed that 8 and 15 polymorphism bands were amplified respectively by using primer BOX.The molecular weight of clust bands ranged from 200 bp to 3000bp, and most of them were 500bp to 2800bp.The amplified polymorphism bands were not distinct by using primer ERIC,showed that it was not adapted to a analyze the genetic diversit of Cmm.Analysis of fingerprints,all strains of Cmm were clustered into 7 groups at a level of 82%similarity,includedâ… group,â…¡group,â…¢group.â…£group,â…¤group.â…¥group andâ…¦group,most of strains were classified intoâ…¥group. It showed abundant DNA polymorphism and diversity and genetic variation of Cmm gained from whole country.The study indicated that it was related to the geo-graphical origin of the pathogen in a certain degree,but it has no relation on year of isolation,and it proved that the bacterial canker of tomato is an soil-borne disease.