| 双重PCR技术检测马铃薯环腐病菌和黑胫病菌方法的建立 |
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| 引用本文： | 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. |
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| 作者姓名： | 韩广涛 杨志辉 朱杰华 赵冬梅 韩彦卿 |
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| 作者单位： | 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 |
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| 基金项目： | 现代农业产业技术体系建设专项资金项目 |
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| 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。
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| 关 键 词： | 马铃薯 环腐病菌 黑胫病菌 双重PCR 分子检测 |
| 收稿时间： | 2011-02-14 |
Development of Duplex PCR Assay for Detection of Clavibacter michiganensis subsp.sepedonicus and Pectobacterium atroseptica from Potato |
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| 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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| Authors: | HAN Guang-tao YANG Zhi-hui ZHU Jie-hua ZHAO Dong-mei HAN Yan-qing |
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| Institution: | HAN Guang-tao1,YANG Zhi-hui1,ZHU Jie-hua1,ZHAO Dong-mei1,HAN Yan-qing2(1College of Plant Protection,Agricultural University of Hebei/Control Centre of Plant Pathogens and Plant Pests of Hebei Province,Baoding 071001,Hebei,2College of Agriculture and Biotechnology,China Agricultural University,Beijing 100193) |
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| 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. |
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| Keywords: | potato Clavibacter michiganensis subsp sepedonicus Pectobacterium atroseptica duplex PCR molecular detection |
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