番茄溃疡病菌的富集和快速分子生物学检测方法研究生物化学与分子生物学专业论文.docxVIP

兰趔太堂殛±堂僮途塞Pathogen 兰趔太堂殛±堂僮途塞 Pathogen Enrichment and Rapid Molecular Biology Detection Methods for Tomato Bacterial Canker Pathogens，Clavibacter michiganensis subsp． michiganensis Abstract Objective：Tomato bacterial canker is the most important bacterial diseases which caused substantial economic losses in the worldwide．Seed is the main long—distance spread pathway．A small amount of bacteria in seed can lead to a large—scale prevalence，Seed born Clavibacter michiganensis subsp．michiganensis(Cmm)is difficult to detect by nomal methods because of its very low rate of infected—seed． Therefore，it is significance to prevent its invasion and spread by establishing all accurate，rapid and sensitive method for Cmm detection． Methods：Comparison of several methods for the detection of Cmm in bacterial suspensions and experimentally contaminated seed samples．These methods including immunological method such嬲ELISA,molecular biology methods such as Direct．．PCR and Nested··PCR,as well as the methods of pathogen enrichment combined with PCR such as BIO—PCR and nitrocellulose membrane capture PCR，the methods of pathogen concentration combined with PCR such as membrane filter PCR， immunocapture PCR(IC—PCR)and immunomagnetic separation PCR(IMS-PCR)． Then,We evaluate the sensitivity,testing period，stability and the cost of these Results：The immunological method and molecular biology methods such aus ELLS八 Direct-·PCR and Nested·-PCR were performed in bacterial suspensions and experimentally contaminated seed samples．These results were compared with the methods of pathogen enrichment or concentration combined with PCR such as nitrocellulose membrane capture PCR，BIO—PCR，membrane filter PCR，IC-PCR and IMS．PCR．ELISA detection limit is more than 106CFU／ml of pathogen in all assayed samples．The detection sensitivity of Direct．PCR is 104CFU／ml and 106CFU／ml in bacterial suspensions and experimentally contaminated seed samples，respectively． Nested．PCR arrived 102CFU／ml and 104CFU／ml in