基于RAA-CRISPR/Cas12a-LFD的杜英疫病菌可视化检测技术的建立  

Establishment of RAA-CRISPR/Cas12a-LFD-based visual detection technology of Pseudocryphonectria elaeocarpicola

作　　者：黄灿 吴浩雨 熊典广 黄华毅 田呈明[1] Huang Can;Wu Haoyu;Xiong Dianguang;Huang Huayi;Tian Chengming(School of Forestry,Beijing Forestry University,Beijing 100083,China;Guangdong Academy of Forestry,Guangzhou 510520,Guangdong,China)

机构地区：[1]北京林业大学林学院,北京100083 [2]广东省林业科学研究院,广东广州510520

出　　处：《北京林业大学学报》2025年第4期10-20,共11页Journal of Beijing Forestry University

基　　金：广东省林业科技创新项目(2020KJCX004、2023KJCX022)。

摘　　要：【目的】由杜英生假隐丛赤壳菌引起的杜英疫病是杜英属植物上新发现的一种危害严重的枝干病害。目前,该病的研究仍处于起步阶段,尚缺乏有效的防控措施。本文旨在建立一种快速、可视化的杜英疫病菌检测方法,为该病害的早期监测和预警提供重要技术支持。【方法】以杜英疫病菌PsGti1基因为靶标,采用重组酶辅助扩增(RAA)反应特异性扩增靶标基因,并利用CRISPR/Cas12a体系切割靶标和荧光探针,最后利用侧向流试纸条(LFD)实现杜英疫病菌的可视化检测。【结果】(1)筛选获得针对杜英疫病菌PsGti1基因RAA扩增反应的最优引物对。(2)当Cas12a与CrRNA浓度配比分别为1μmol/L和0.125μmol/L时CRISPR/Cas12a切割反应体系的效果最好。(3)建立了基于RAA-CRISPR/Cas12a-LFD的可视化检测体系,在37℃恒温反应条件下可快速检测杜英疫病菌,检测灵敏度为50 fg/μL(以gDNA为模板)和20 pg/μL(以PsGti1_T-vector为模板)。【结论】本研究建立的杜英疫病菌RAA-CRISPR/Cas12a-LFD检测体系具有反应温度易达到、高特异性、高灵敏度和易操作等优点,适合在野外或缺乏实验室检测设备的场景下进行杜英疫病菌的检测。[Objective]Elaeocarpus spp.stem blight is a newly discovered branch disease on Elaeocarpus spp.caused by Pseudocryphonectria elaeocarpicola,for which research is still in its infancy and lacks effective prevention and control measures.This paper aims to establish a rapid and visualized method for the detection of P.elaeocarpicola,which provides an important technical support for the early monitoring and early warning of the disease.[Method]In this study,we utilized PsGti1 gene of P.elaeocarpicola as a target,specifically amplified the target gene by recombinase aided amplification(RAA)reaction,and cut the target and fluorescent probe by CRISPR/Cas12a system,and finally visualized the detection of P.elaeocarpicola by lateral flow dipstick(LFD).[Result](1)In this study,the optimal primer pairs for the RAA amplification reaction of PsGti1 gene of P.elaeocarpicola were screened and obtained.(2)The CRISPR/Cas12a reaction system worked best when Cas12a and CrRNA concentration ratios were 1 and 0.125μmol/L,respectively.(3)A RAA-CRISPR/Cas12a-LFD visualization detection system was established,which can realize the rapid detection of P.elaeocarpicola under the reaction condition of constant temperature at 37℃,and the sensitivity of detection was 50 fg/μL(gDNA as template)and 20 pg/μL(PsGti1_T-vector as template),respectively.[Conclusion]The RAA-CRISPR/Cas12a-LFD detection system for P.elaeocarpicola established in this study has the advantages of easy-to-reach reaction temperature,high specificity,high sensitivity and ease of operation,which makes it suitable for carrying out the detection of P.elaeocarpicola in the field or in scenarios lacking laboratory testing equipment.

关 键 词：杜英疫病菌 重组酶辅助扩增(RAA) CRISPR/Cas12a 侧向流试纸条(LFD) 可视化检测 

分 类 号：S763.1[农业科学—森林保护学]