| 摘要: |
| 【目的】大麦坚黑粉菌（Ustilago hordei）和裸黑粉菌（Ustilago nuda）是引起青藏高原青稞黑穗病的主要病原菌，建立快速、简便、特异性强的大麦坚黑粉菌和裸黑粉菌环介导等温扩增技术（LAMP）检测体系，检测青稞种子的带菌量，再依据带菌程度进行播前种子处理，是控制青稞黑穗病的重要技术手段。【方法】根据大麦坚黑粉菌（Ustilago hordei）和裸黑粉菌（Ustilago nuda）的ITS基因序列与其他对照菌株同源性低的区段设计并获得有效引物1组，建立LAMP检测体系，并选取影响LAMP反应体系的5个主要因素dNTPs、甜菜碱、镁离子、Bst DNA聚合酶及温度进行单因素试验对其进行优化，最后对优化后的LAMP检测体系进行灵敏度和特异性检测。【结果】建立了青稞黑穗病菌的LAMP检测体系，其优化后dNTPs浓度为1.8 mmol/L、甜菜碱浓度为0.8 mmol/L、镁离子浓度为10 mmol/L、Bst DNA聚合酶浓度为320 U/mL，反应温度为65 ℃。应用该体系在45 min内能检测出2×10-4 ng/μL的裸黑粉菌，在60 min内能检测出2×10-4 ng/μL的大麦坚黑粉菌，即在60 min内可检测出2×10-4 ng/μL的大麦坚黑粉菌和裸黑粉菌。建立的青稞黑穗病菌LAMP检测体系对大麦坚黑粉菌和祼黑粉菌具有特异性。【结论】建立了青稞黑穗病菌的 LAMP检测体系，该体系能在1 h内检测出2×10-4 ng/μL的裸黑粉菌和大麦坚黑粉菌。 |
| 关键词: 大麦坚黑粉菌 裸黑粉菌 青稞黑穗病 环介导等温扩增技术 |
| DOI： |
| 分类号: |
| 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 for barley smut |
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YANG Fan,HU Zhangwei,YAN Haohao,et al
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| Abstract: |
| 【Objective】Ustilago hordei and Ustilago nuda are main pathogens that cause smut disease on the Qinghai-Tibet Plateau.The establishment of loop mediated isothermul amplification (LAMP) detection system of U.hordei and U.nuda to detect fungi amount carried by barley seeds with high efficiency and specificity and seed treatments before sowing provided an important technical method to control barley smut.【Method】One set of primers were designed based on ITS gene regions of U.hordei and U.nuda that had low homology with other control strains.Five main factors in the LAMP reaction system including dNTPs,concentrations of betaine,Mg2+ and Bst DNA polymerase,and annealing temperature were selected and optimized to increase detection sensitivity and specificity.【Result】The LAMP detection system of barley smut was established successfully.The optimized reaction conditions included dNTPs 1.8 mmol/L,betaine 0.8 mmol/L,Mg2+ 10 mmol/L,Bst DNA polymerase 320 U/mL and annealing temperature at 65 ℃.Under such conditions,2×10-4 ng/μL U.nuda and U.hordei were detected within 45 and 60 min,respectively.The LAMP system had specificity to U.nuda and U.hordei.【Conclusion】The established LAMP method can be used to detect U.nuda and U.hordei in highland barley at the level of 2×10-4 ng/μL within 1 h.This study provides foundation for development of other barley seed borne fungus detection methods. |
| Key words: Ustilago hordei Ustilago nuda barley smut loop-mediated isothermal amplification (LAMP) |
