基于RPA-CRISPR/Cas12a的小麦矮腥黑粉菌快速检测技术OA
RPA-CRISPR/Cas12a-based rapid detection for Tilletia controversa
小麦矮腥黑穗病(wheat dwarf bunt disease,DB)是由小麦矮腥黑粉菌 Tilletia controversa(TCK)侵染引发的重大检疫性病害.实现TCK的精准快速检测,对口岸检疫与病害防控工作至关重要.本研究以 TCK 基因组(LWDE02001036.1)上保守性较高的基因片段为靶标,建立了重组酶聚合酶等温扩增技术(RPA)结合 CRISPR/Cas12a 的快速可视化检测体系.该体系在 37℃恒温条件下,20 min 内即可完成目标病原菌检测,对 TCK 基因组 DNA 和质粒检测灵敏度分别达 200 fg/μL 和101 copy/μL,对冬孢子检测下限为 40 个.检测结果可通过核酸试纸条颜色反应直观读取.该体系具备特异、灵敏和快速等优势,为基层口岸 TCK 检疫提供了技术支撑.
Wheat dwarf bunt disease(DB)is a major quarantinable disease caused by the infection of Tilletia controversa(TCK).Accurate and rapid detection of TCK is crucial for port quarantine and disease prevention and control.In this study,a rapid visual detection system combining recombinase polymerase amplification(RPA)with CRISPR/Cas12a was established by targeting a highly conserved gene fragment on the TCK genome(accession No.LWDE02001036.1).This system could complete the detection of the target pathogen within 20 minutes under a constant temperature of 37℃,with the detection sensitivities of 200 fg/μL and 10¹ copies/μL for TCK genomic DNA and recombinant plasmids,respectively,and the minimum detectable limit of 40 teliospores.The detection results can be directly read by the color reaction of nucleic acid test strips.With the advantages of high specificity,high sensitivity and rapidity,this system provides technical support for TCK quarantine at grassroots ports.
何畅;崇钰;黄洁;李媛;高利;王保通;李强
西北农林科技大学植物保护学院 作物抗逆与高效生产全国重点实验室,陕西 杨凌 712100西北农林科技大学植物保护学院 作物抗逆与高效生产全国重点实验室,陕西 杨凌 712100西北农林科技大学植物保护学院 作物抗逆与高效生产全国重点实验室,陕西 杨凌 712100西北农林科技大学植物保护学院 作物抗逆与高效生产全国重点实验室,陕西 杨凌 712100中国农业科学院植物保护研究所,北京 100193西北农林科技大学植物保护学院 作物抗逆与高效生产全国重点实验室,陕西 杨凌 712100西北农林科技大学植物保护学院 作物抗逆与高效生产全国重点实验室,陕西 杨凌 712100
快速检测重组酶聚合酶等温扩增CRISPR/Cas12a检测体系小麦矮腥黑粉菌
rapid detectionrecombinase polymerase amplification(RPA)CRISPR/Cas12a detection systemTilletia controversa(TCK)
《菌物学报》 2026 (9)
95-105,11
科技部对发展中国家援助项目(KY202002018)陕西省重点研发计划(2025NC-YBXM-049) This work was supported by the Ministry of Science and Technology Aid Project to Developing Countries(KY202002018)and the Key Research and Development Program of Shaanxi Province(2025NC-YBXM-049).
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