基于实时荧光LAMP的马铃薯黑胫病菌快速可视化检测方法的建立 Click here to download the full text Citation：王海源,王玥,马永强.基于实时荧光LAMP的马铃薯黑胫病菌快速可视化检测方法的建立.Journal of Plant Protection,2026,53(4):1010-1018 Hits: Download times: Author Name Affiliation E-mail Wang Haiyuan  Qinghai Provincial Key Laboratory of Integrated Pest Management in Agriculture, College of Agriculture and Forestry, Qinghai University, Xining 810016, Qinghai Province, China    Wang Yue  Qinghai Provincial Key Laboratory of Integrated Pest Management in Agriculture, College of Agriculture and Forestry, Qinghai University, Xining 810016, Qinghai Province, China    Ma Yongqiang  Qinghai Provincial Key Laboratory of Integrated Pest Management in Agriculture, College of Agriculture and Forestry, Qinghai University, Xining 810016, Qinghai Province, China  mayongqiang_163@163.com  中文摘要:为构建马铃薯黑胫病的主要病原菌马铃薯黑腐果胶杆菌Pectobacterium atrosepticum的高效分子检测方法，基于环介导等温扩增（loop-mediated isothermal amplification，LAMP）技术，以黑腐果胶杆菌gyrB基因的保守片段设计特异性LAMP引物，通过反应体系的筛选和优化，建立马铃薯黑胫病菌快速检测方法。结果表明：LAMP检测体系的最佳反应条件为Mg2+ 4 mmol/L、dNTP1.6 mmol/L、Bst 2.0 DNA聚合酶6.4 U/mL、甜菜碱0.3 mol/L、反应温度61℃、反应时间40 min；LAMP反应体系对黑腐果胶杆菌的检测具有良好特异性，与其他供试病原菌均无交叉反应，对基因组DNA的最低检测限度为6.4 pg/μL，灵敏度是常规实时荧光定量PCR方法的25倍。利用该检测方法对田间疑似感染黑胫病样本检测的结果与常规PCR方法检测结果一致，表明所建立的可视化LAMP检测方法适用于黑腐果胶杆菌引发的马铃薯黑胫病的早期诊断和田间病情监测。 中文关键词:马铃薯  黑胫病  LAMP检测  黑腐果胶杆菌   Establishment of rapid detection method for potato blackleg disease based on LAMP real-time fluorescence and visualization Author Name Affiliation E-mail Wang Haiyuan  Qinghai Provincial Key Laboratory of Integrated Pest Management in Agriculture, College of Agriculture and Forestry, Qinghai University, Xining 810016, Qinghai Province, China    Wang Yue  Qinghai Provincial Key Laboratory of Integrated Pest Management in Agriculture, College of Agriculture and Forestry, Qinghai University, Xining 810016, Qinghai Province, China    Ma Yongqiang  Qinghai Provincial Key Laboratory of Integrated Pest Management in Agriculture, College of Agriculture and Forestry, Qinghai University, Xining 810016, Qinghai Province, China  mayongqiang_163@163.com  Abstract:In order to construct an efficient molecular detection method for Pectobacterium atrosepticum, the main pathogen of potato blackleg disease, a rapid detection method was established based on loop-mediated isothermal amplification (LAMP). The specific LAMP primers were designed based on the conserved region of gyrB gene in P. atrosepticum, and a rapid detection method for this fungus was established by screening and optimizing the reaction system. The results showed that the optimal reaction conditions for the LAMP detection system were determined as follows: 4 mmol/L Mg2+, 1.6 mmol/L dNTPs, 6.4 U/mL Bst 2.0 DNA polymerase, 0.3 mol/L betaine, a reaction temperature of 61 ℃, and a reaction time of 40 min. The LAMP reaction system had high specificity for the detection of P. atrosepticum, and had no cross-reaction with other pathogens tested. The minimum detection limit of the genomic DNA was 6.4 pg/μL, and the sensitivity was 25-fold that of the conventional qPCR method. The results of the detection of suspected blackleg samples in the field by this detection method were consistent with those of the conventional PCR method, indicating that the established visual LAMP detection method was suitable for the early diagnosis and field monitoring of potato blackleg disease caused by P. atrosepticum. keywords:potato  potato black leg  LAMP detection  Pectobacterium atrosepticum View Full Text  View/Add Comment  Download reader

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