双重PCR技术检测马铃薯环腐病菌和黑胫病菌方法的建立

doi:10.3864/j.issn.0578-1752.2011.20.008

中国农业科学 ›› 2011, Vol. 44 ›› Issue (20): 4199-4206.doi: 10.3864/j.issn.0578-1752.2011.20.008

双重PCR技术检测马铃薯环腐病菌和黑胫病菌方法的建立

韩广涛, 杨志辉, 朱杰华, 赵冬梅, 韩彦卿

1.河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术研究中心
2.中国农业大学农学与生物技术学院

收稿日期:2011-02-14 出版日期:2011-10-15 发布日期:2011-05-03
通讯作者: 通信作者朱杰华，Tel：0312-7528175；E-mail：zhujiehua356@yahoo.com.cn
作者简介:韩广涛，Tel：0312-7528587
基金资助:
现代农业产业技术体系建设专项资金（CARS-10-P12）

Development of Duplex PCR Assay for Detection of Clavibacter michiganensis subsp. sepedonicus and Pectobacterium atroseptica from Potato

HAN Guang-Tao, YANG Zhi-Hui, ZHU Jie-Hua, ZHAO Dong-Mei, HAN Yan-Qing

1.河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术研究中心
2.中国农业大学农学与生物技术学院

Received:2011-02-14 Online:2011-10-15 Published:2011-05-03

摘要/Abstract

摘要： 【目的】利用双重PCR技术快速检测马铃薯环腐病菌（Clavibacter michiganensis subsp. sepedonicus）和黑胫病菌（Pectobacterium atroseptica）。【方法】根据GenBank上发表的马铃薯环腐病菌pCS1质粒上纤维素酶A基因序列，对比近缘种及马铃薯上几种重要病原菌的核苷酸序列，设计并合成了1对特异性引物CMS1/CMS2，将设计的引物与已发表的PCR检测马铃薯黑胫病菌特异性引物ECA1f/ECA2r结合，经过条件优化后，建立了双重PCR体系。【结果】利用引物CMS1/CMS2扩增出了1条913 bp的马铃薯环腐病菌特异性条带。检测灵敏度在DNA水平上达100 fg•μL-1，在细菌数上达105 CFU•mL-1。利用双重PCR体系对马铃薯环腐病菌和黑胫病菌进行扩增，可获得913和690 bp的2条特异性条带。检测灵敏度在DNA水平上达600 fg•μL-1，在细菌数上达5×105 CFU•mL-1。【结论】成功建立了双重PCR检测马铃薯环腐病菌和黑胫病菌技术体系，该技术能够同时快速可靠地检测出马铃薯环腐病菌和黑胫病菌。

关键词:

马铃薯, 环腐病菌, 黑胫病菌, 双重PCR, 分子检测

Abstract: 【Objective】The objective of this study is to develop a duplex PCR system to simultaneously and reliably detect Clavibacter michiganensis subsp. sepedonicus and Pectobacterium atroseptica. 【Method】 Choosing the cellulose A gene sequence encoded by the native plasmid pCS1 of C. michiganensis subsp. sepedonicus which was published on the GenBank and comparing it with the nucleotide sequence of closely-related species and some pathogens of potato, a specific pair of primers, CMS1/CMS2, was designed and synthesized. A duplex PCR system had been established under the optimized PCR parameters using the combining primers CMS1/CMS2 and ECA1f/ECA2r which was a specific pair of PCR primers to detect P. atroseptica. 【Result】 Using CMS1/CMS2 primers, a single unique PCR band of 913 bp was amplified from C. michiganensis subsp. sepedonicus. The detection sensitivity was 100 fg•μL-1 of DNA and 105 CFU•mL-1 of bacteria. Under the duplex PCR system, the 913 and 690 bp PCR bands from P. atroseptica could be specifically amplified. The detection sensitivity was 600 fg•μL-1 of DNA and 5×105 CFU•mL-1 of bacteria.【Conclusion】The duplex PCR system could simultaneously and rapidly detect the two pathogens.

Key words: potato, Clavibactermichiganensissubsp.sepedonicus, Pectobacteriumatroseptica, duplexPCR, moleculardetection

韩广涛, 杨志辉, 朱杰华, 赵冬梅, 韩彦卿. 双重PCR技术检测马铃薯环腐病菌和黑胫病菌方法的建立[J]. 中国农业科学, 2011, 44(20): 4199-4206.

HAN Guang-Tao, YANG Zhi-Hui, ZHU Jie-Hua, ZHAO Dong-Mei, HAN Yan-Qing. Development of Duplex PCR Assay for Detection of Clavibacter michiganensis subsp. sepedonicus and Pectobacterium atroseptica from Potato[J]. Scientia Agricultura Sinica, 2011, 44(20): 4199-4206.

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双重PCR技术检测马铃薯环腐病菌和黑胫病菌方法的建立

Development of Duplex PCR Assay for Detection of Clavibacter michiganensis subsp. sepedonicus and Pectobacterium atroseptica from Potato

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