双重PCR和竞争性PCR检测马铃薯环腐病菌和黑胫病菌技术体系建立
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摘要
马铃薯是世界上仅次于水稻、小麦和玉米的第四大粮食作物,我国为世界第一大马铃薯生产国。马铃薯环腐病(Clavibacter michiganensis subsp. Sepedonicus, Cms)和黑胫病(Pectobacterium atroseptica, Eca)是影响马铃薯生产的两个重要的种薯传播细菌性病害,能够快速、准确、灵敏、可靠地检测这两种病害对降低种薯带菌减轻病害发生具有重要意义。本研究建立了双重PCR检测黑胫病菌和环腐病菌技术和竞争性PCR定量检测黑胫病菌技术。
主要研究结果如下:
1.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细菌的核苷酸序列,设计并合成了一对特异性引物CMS1/CMS2,利用引物CMS1/CMS2扩增出了一条913bp的马铃薯环腐病菌特异性条带,而马铃薯环腐病菌近缘种和马铃薯上的重要病原菌均未扩增出任何条带,特异性很高。检测灵敏度在DNA水平上达100fg/μL,在细菌数量上达10~5CFU/mL。
2.将设计的引物CMS1/CMS2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,成功建立了双重PCR技术体系。利用该双重PCR体系对马铃薯环腐病菌和黑胫病菌进行扩增,可获得913bp和690bp的2条特异性条带,而15种对照菌株均未扩增出任何条带,检测灵敏度在DNA水平上达600fg/μL,在细菌数量上达5×10~5CFU/mL。应用该体系对患环腐病和(或)黑胫病的薯块进行了检测,能够检测到环腐病菌和(或)黑胫病菌的存在。实现了同时对马铃薯环腐病菌和黑胫病菌的快速可靠检测。
3.应用黑胫病菌特异性引物ECA1f/ECA2r扩增黑胫病菌DNA,将扩增产物克隆测序。根据扩增产物序列和引物ECA1f/ECA2r序列,设计了新引物ECA3f,与ECA2r组合扩增黑胫病菌DNA,制备竞争性模板。将竞争性模板插入pEASY-T1克隆载体,导入Trans1-T1感受态细胞进行克隆操作,构建内参照菌(E.coli 3f)。将已知数量的E.coli 3f加入到含Eca的无菌水或马铃薯皮提取物中,通过比较马铃薯黑胫病菌与E.coli 3f的扩增产物间的亮度比率能够估计马铃薯黑胫病菌的数量。
Potato is the fourth food crop in the world, which is behind rice, wheat and corn. China is the world’s biggest producer of potato. Potato ring rot (Clavibacter michiganensis subsp. Sepedonicus, Cms) and Potato blackleg (Pectobacterium atroseptica ,Eca) are the two important seed transmitted bacterial disease which impact on the potato production. Fast, accurate, sensitive and reliable detection of these two diseases is of great significance on reducing the percentage of infected seed tubers and the occurrence of the diseases. A duplex PCR assay for detecting of Clavibacter michiganensis subsp. sepedonicus and Pectobacterium atroseptica and a competitive PCR assay for quantification of Pectobacterium atroseptica were established. The results were as follows.
1. 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. Using CMS1/CMS2 primers, a single unique PCR band of 913 bp was amplified from C. michiganensis subsp. sepedonicus. The detection sensitivity was 100fg/μL of DNA and 10~5 CFU/mL of bacteria.
2. A duplex PCR technology 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 Pectobacterium atroseptica. Under the duplex PCR system, the 913 bp PCR band from Clavibacter michiganensis subsp. sepedonicus and 690 bp PCR band from P. atroseptica could be specifically amplified. The detection sensitivity was 600fg/μL of DNA and 5×10~5CFU/mL of bacteria. The duplex PCR technology also can be used to detect the tuber suffered from Clavibacter michiganensis subsp. sepedonicus and(or) Pectobacterium atroseptica and achieved good results. The two pathogens could be simultaneously and rapidly detected by the duplex PCR system.
3. Eca genomic DNA was amplified using Eca specific primers ECA1f/ECA2r. The amplification product was cloned and sequenced. Using the DNA sequence of the amplification product and the sequences of specific primers ECA1f/ECA2r, a new forward primer, ECA3f, was designed and synthesized. Eca genomic DNA was amplified using new specific primers ECA3f/ECA2r, the amplification product is competitor PCR template. The competitor template was cloned into pEASY-T1 Clong Vector and transformed into Trans1-T1 competent cell, and internal control (E.coli 3f) was constructed for competitive PCR. Predetermined numbers of E.coli 3f were added to sterile water or potato peel extract both of which have Eca, and Eca numbers estimated by comparing the ratio of products generated Eca and E.coli 3f following PCR.
主要研究结果如下:
1.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细菌的核苷酸序列,设计并合成了一对特异性引物CMS1/CMS2,利用引物CMS1/CMS2扩增出了一条913bp的马铃薯环腐病菌特异性条带,而马铃薯环腐病菌近缘种和马铃薯上的重要病原菌均未扩增出任何条带,特异性很高。检测灵敏度在DNA水平上达100fg/μL,在细菌数量上达10~5CFU/mL。
2.将设计的引物CMS1/CMS2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,成功建立了双重PCR技术体系。利用该双重PCR体系对马铃薯环腐病菌和黑胫病菌进行扩增,可获得913bp和690bp的2条特异性条带,而15种对照菌株均未扩增出任何条带,检测灵敏度在DNA水平上达600fg/μL,在细菌数量上达5×10~5CFU/mL。应用该体系对患环腐病和(或)黑胫病的薯块进行了检测,能够检测到环腐病菌和(或)黑胫病菌的存在。实现了同时对马铃薯环腐病菌和黑胫病菌的快速可靠检测。
3.应用黑胫病菌特异性引物ECA1f/ECA2r扩增黑胫病菌DNA,将扩增产物克隆测序。根据扩增产物序列和引物ECA1f/ECA2r序列,设计了新引物ECA3f,与ECA2r组合扩增黑胫病菌DNA,制备竞争性模板。将竞争性模板插入pEASY-T1克隆载体,导入Trans1-T1感受态细胞进行克隆操作,构建内参照菌(E.coli 3f)。将已知数量的E.coli 3f加入到含Eca的无菌水或马铃薯皮提取物中,通过比较马铃薯黑胫病菌与E.coli 3f的扩增产物间的亮度比率能够估计马铃薯黑胫病菌的数量。
Potato is the fourth food crop in the world, which is behind rice, wheat and corn. China is the world’s biggest producer of potato. Potato ring rot (Clavibacter michiganensis subsp. Sepedonicus, Cms) and Potato blackleg (Pectobacterium atroseptica ,Eca) are the two important seed transmitted bacterial disease which impact on the potato production. Fast, accurate, sensitive and reliable detection of these two diseases is of great significance on reducing the percentage of infected seed tubers and the occurrence of the diseases. A duplex PCR assay for detecting of Clavibacter michiganensis subsp. sepedonicus and Pectobacterium atroseptica and a competitive PCR assay for quantification of Pectobacterium atroseptica were established. The results were as follows.
1. 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. Using CMS1/CMS2 primers, a single unique PCR band of 913 bp was amplified from C. michiganensis subsp. sepedonicus. The detection sensitivity was 100fg/μL of DNA and 10~5 CFU/mL of bacteria.
2. A duplex PCR technology 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 Pectobacterium atroseptica. Under the duplex PCR system, the 913 bp PCR band from Clavibacter michiganensis subsp. sepedonicus and 690 bp PCR band from P. atroseptica could be specifically amplified. The detection sensitivity was 600fg/μL of DNA and 5×10~5CFU/mL of bacteria. The duplex PCR technology also can be used to detect the tuber suffered from Clavibacter michiganensis subsp. sepedonicus and(or) Pectobacterium atroseptica and achieved good results. The two pathogens could be simultaneously and rapidly detected by the duplex PCR system.
3. Eca genomic DNA was amplified using Eca specific primers ECA1f/ECA2r. The amplification product was cloned and sequenced. Using the DNA sequence of the amplification product and the sequences of specific primers ECA1f/ECA2r, a new forward primer, ECA3f, was designed and synthesized. Eca genomic DNA was amplified using new specific primers ECA3f/ECA2r, the amplification product is competitor PCR template. The competitor template was cloned into pEASY-T1 Clong Vector and transformed into Trans1-T1 competent cell, and internal control (E.coli 3f) was constructed for competitive PCR. Predetermined numbers of E.coli 3f were added to sterile water or potato peel extract both of which have Eca, and Eca numbers estimated by comparing the ratio of products generated Eca and E.coli 3f following PCR.
引文
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