双重PCR方法检测检疫性细菌洋葱腐烂病菌和菊基腐病菌

doi:10.3969/j.issn.1004-1524.2018.03.11

浙江农业学报 ›› 2018, Vol. 30 ›› Issue (3): 426-431.DOI: 10.3969/j.issn.1004-1524.2018.03.11

双重PCR方法检测检疫性细菌洋葱腐烂病菌和菊基腐病菌

杨万风¹, 刘艳², 陆辰晨¹, 邵沛泽¹, 谌运清¹, 赵文军³

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

收稿日期:2017-07-19 出版日期:2018-03-20 发布日期:2018-03-21
作者简介:杨万风(1979—),男,江苏兴化人,硕士,主要从事检疫性细菌致病性研究。E-mail: ywf307@163.com
基金资助:
江苏省科技计划项目(2017KJ29)

Duplex PCR detection of Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi

YANG Wanfeng¹, LIU Yan², LU Chenchen¹, SHAO Peize¹, CHEN Yunqing¹, ZHAO Wenjun³

1. Lianyungang Entry-Exit Inspection and Quarantine Bureau, Lianyungang 222042, China;
2. Lianyungang Academy of Agricultural Sciences, Lianyungang 222001, China;
3. Chinese Academy of Inspection and Quarantine, Beijing 100029, China

Received:2017-07-19 Online:2018-03-20 Published:2018-03-21

摘要/Abstract

摘要： 为建立同步检测检疫性细菌洋葱腐烂病菌和菊基腐病菌的双重PCR方法,根据GenBank上公布的洋葱腐烂病菌ITS序列设计1对特异性引物B3/B6,将设计的引物与已发表的检测菊基腐病菌特异性引物ADE1/ADE2结合,经过条件优化,建立了双重PCR反应体系,并进行特异性和灵敏度验证。应用双重PCR体系能从洋葱腐烂病菌和菊基腐病菌基因组DNA以及人工带菌样品中扩增到特异性条带。结果表明,建立的双重PCR检测方法能同时检测出洋葱腐烂病菌和菊基腐病菌,可应用于口岸进口郁金香种球2种检疫性细菌的检测。

关键词: 洋葱腐烂病菌, 菊基腐病菌, 双重PCR, 检测方法

Abstract: The objective of this study is to establish a duplex PCR technology for simultaneously detecting Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi. Based on the 16S-23S rRNA intergenic transcribed spacer sequence of B. gladioli pv. alliicola in GenBank, the primers B3/B6 were designed. The duplex PCR assay had been developed using the combining primers B3/B6 and ADE1/ADE2 which were specific primers to detect E. chrysanthemi. The reaction conditions were optimized and the specificity and sensitivity of the duplex PCR were tested. The expected DNA fragment could be specifically amplified from the genomic DNA of B. gladioli pv. alliicola and E. chrysanthemi. Specificity was confirmed using the duplex PCR assay to detect B. gladioli pv. alliicola and E. chrysanthemi in the artificially inoculated tulip leaf samples. The duplex PCR developed in this study can be used in detecting the two pathogens for imported tulip bulbs quarantine.

Key words: Burkholderia gladioli pv. alliicola, Erwinia chrysanthemi, duplex PCR, detection

中图分类号:

S436.33

杨万风, 刘艳, 陆辰晨, 邵沛泽, 谌运清, 赵文军. 双重PCR方法检测检疫性细菌洋葱腐烂病菌和菊基腐病菌[J]. 浙江农业学报, 2018, 30(3): 426-431.

YANG Wanfeng, LIU Yan, LU Chenchen, SHAO Peize, CHEN Yunqing, ZHAO Wenjun. Duplex PCR detection of Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi[J]. , 2018, 30(3): 426-431.

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双重PCR方法检测检疫性细菌洋葱腐烂病菌和菊基腐病菌

Duplex PCR detection of Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi

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