巢式PCR检测菜豆细菌性萎蔫病菌

浙江农业学报

巢式PCR检测菜豆细菌性萎蔫病菌

（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）

出版日期:2015-07-25 发布日期:2015-08-03

Detection of Curtobacterium flaccumfaciens pv. flaccumfaciens using nested PCR#br#

（1 Lianyungang Entry\|Exit Inspection and Quarantine Bureau， Lianyungang 222042，China; 2 Lianyungang Academy of Agricultrual Sciences， Lianyungang 222001，China; 3 Chinese Academy of Inspection and Quarantine， Beijing 100029，China）

Online:2015-07-25 Published:2015-08-03

摘要/Abstract

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CffF11，与引物CffFOR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1×103 cfu·mL-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1 h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。

关键词: 菜豆细菌性萎蔫病菌, 巢式PCR, 检测方法

Abstract: With the purpose of fast detecting of Curtobacterium flaccumfaciens pv. flaccumfaciens in imported soybean， we established nested PCR with the reported primer CffFOR and the primer CffF11 based on the REP\|PCR sequenced results of C. flaccumfaciens pv. flaccumfaciens and testified its specificity and sensitivity. In specificity assays， only the samples of C. flaccumfaciens pv. flaccumfaciens were positive， while the other pathogens were negative. The detecting sensitivity of the bacterial suspension using nested PCR was 31×103 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 detected 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 established 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.

Key words: Curtobacterium flaccumfaciens pv. flaccumfaciens, nested PCR, detection

杨万风1，刘艳2,刘翔1,邵沛泽1,谌运清1,赵文军3. 巢式PCR检测菜豆细菌性萎蔫病菌[J]. 浙江农业学报.

YANG Wan\|feng1， LIU Yan2， LIU Xiang1， SHAO Pei\|ze1， CHEN Yun\|qing1， ZHAO Wen\|jun3. Detection of Curtobacterium flaccumfaciens pv. flaccumfaciens using nested PCR#br#[J]. .