| 摘要: |
| 【目的】建立青稞条纹病的环介导等温扩增（loop-mediated isothermal amplification,LAMP）技术检测体系，为青稞种子带菌检测及其条纹病的有效防控提供参考。【方法】根据麦类核腔菌（Pyrenophora graminea）的ITS-rDNA序列区间设计5组引物，从中筛选特异性引物，并通过设置60，61，62，63，64和65 ℃ 6个温度梯度来优化LAMP反应体系的温度，以建立高效检测麦类核腔菌的LAMP技术体系；分别以麦类核腔菌、大麦坚黑粉菌、裸黑粉菌、禾生指葡孢霉、细链格孢和禾谷镰孢菌基因组DNA为模板进行LAMP扩增反应，对该体系的特异性进行检测；以310，31，3.1，0.31，0.031和0.003 1 pg/μL的麦类核腔菌基因组DNA为模板，对该体系的灵敏度进行分析；最后利用该体系对肚里黄、柴青1号、1141、北青9号和昆仑15 等5个青稞主栽品种的种子带菌率进行检测。【结果】青稞条纹病LAMP检测体系的特异性引物为引物组2，最佳反应温度63 ℃，反应时间70 min；该检测体系对麦类核腔菌具有良好的特异性，其最低检测限为0.31 pg/μL；肚里黄、柴青1号、1141、北青9号和昆仑15种子的带菌率分别为68.18%，72.73%，100%，77.27%和68.18%。【结论】建立了青稞条纹病LAMP检测体系，该体系能在63 ℃、70 min内检测出青稞种子是否携带条纹病菌。 |
| 关键词: 青稞条纹病 环介导等温扩增 LAMP检测体系 |
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| 基金项目:青海省基础研究计划项目（2020-ZJ-748）；青海省重点实验室项目（2020-ZY-Y11） |
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| Establishment of LAMP detection system of Pyrenophora graminea |
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HU Zhangwei,YANG Fan,ZHANG Xuefei,et al
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| Abstract: |
| 【Objective】To provide a reference for detection of barley seeds and effective prevention and control of barley stripe,this study established a detection system for barley stripe based on loop-mediated isothermal amplification (LAMP) technology.【Method】In this study,five sets of specific primers were designed based on the ITS-rDNA sequences of P. graminea,and one set was screened to optimize reaction temperature based on six gradients (60，61，62，63，64 and 65 ℃).Then,the LAMP system for efficient detection of the pathogen was established.Its specificity was tested based on LAMP amplification of DNA of genes from P.graminea,Ustilago hordei,Ustilago nuda,Dactylobotrys graminicola,Alternaria alternata and Fusarium graminearum.The sensitivity was analyzed by testing genes at levels of 310，31，3.1，0.31，0.031 and 0.003 1 pg/μL.At last,it was used for testing and verifying seeds of five barley varieties.【Result】The established LAMP methods for detecting P. graminea performed optimally at 63 ℃ for 70 min with specific primers of set 2.Under the optimal reaction conditions,this method had good specificity for P. graminea with minimum detection limit of 0.31 pg/μL.The P. gramineacarrying rates of Dulihuang,Chaiqing 1,1141,Beiqing 9 and Kunlun 15 seeds were 68.18%，72.73%，100%，77.27% and 68.18%,respectively.【Conclusion】The established LAMP method can be used to detect the P. graminea in barley seeds within 70 min at 63 ℃. |
| Key words: Pyrenophora graminea loop-mediated isothermal amplification (LAMP) LAMP detection system |
