巢式PCR检测菜豆细菌性萎蔫病菌  

Detection of Curtobacterium flaccumfaciens pv. flaccumfaciens using nested PCR

作　　者：杨万风[1] 刘艳[2] 刘翔[1] 邵沛泽[1] 谌运清[1] 赵文军[3] 

机构地区：[1]连云港出入境检验检疫局,江苏连云港222042 [2]连云港市农业科学院,江苏连云港222001 [3]中国检验检疫科学研究院,北京100029

出　　处：《浙江农业学报》2015年第7期1202-1207,共6页Acta Agriculturae Zhejiangensis

基　　金：国家质检总局科技计划项目(2013IK277)

摘　　要：为了实现对进口大豆样品中菜豆细菌性萎蔫病菌的快速检测,试验根据菜豆细菌性萎蔫病菌的REP-PCR扩增测序结果,设计了1条特异性引物Cff F11,与引物Cff FOR组合,建立了巢式PCR检测方法,并进行特异性和灵敏度验证。结果表明,利用此检测方法对多种参试菌株进行检测时,只有菜豆细菌性萎蔫病菌呈阳性反应,而其他病菌不产生扩增反应;巢式PCR方法检测菜豆细菌性萎蔫病菌菌悬液时,其灵敏度达到3.1×103cfu·m L-1,比常规PCR灵敏度高1 000倍。制备带菌率为0.5%的大豆样品,用巢式PCR方法 1h内就可进行检测,检测时间大大缩短;在对实验室留存的100份进口大豆样品检测时,1份样品扩增到了特异性条带,测序分析结果证明此份大豆样品中携带的病菌为菜豆细菌性萎蔫病菌。本试验所建立的巢式PCR检测方法具有特异性强、灵敏度高、检测时间短并且检测准确率高等特点,可用于口岸菜豆细菌性萎蔫病菌的检测。With the purpose of fast detecting of Curtobacterium flaccumfaciens pv. flaccumfaciens in imported soy- bean, we established nested PCR with the reported primer CffFOR and the primer CffF11 based on the REP-PCR se- quenced results of C. flaccumfaciens pv. flaccumfaciens and testified its specificity and sensitivity. In specificity as- says, only the samples of C. flaccumfaciens pv. flaccumfaciens were positive, while the other pathogens were nega- tive. The detecting sensitivity of the bacterial suspension using nested PCR was 3.1 x 103 cfu-mL-1 which was 1 000 times higher than normal PCR. Moreover, it could detect the pathogen in the soybean sample with 0. 5% infected rate within 1 h, which significantly shortened the testing time. One hundred imported samples in the lab were detec- ted by nested PCR, the specific DNA band could be amplified only from one sample, and the sequence results showed that the soybean sample was indeed infected by C. flaccumfaciens pv. flaccumfaciens. The nested PCR estab- lished in this study has properties of strong specificity, high sensitivity, shorter testing time and accurate result, it can be used in detecting C. flaccumfaciens pv. flaccumfaciens for imported soybean.

关 键 词：菜豆细菌性萎蔫病菌 巢式PCR 检测方法 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]