北京林业大学学报2025，Vol.47Issue(4)：10-20,11.DOI:10.12171/j.1000-1522.20240341
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
摘要
Abstract
[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 0.125 μmol/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Key words
Pseudocryphonectria elaeocarpicola/recombinase-aided amplification(RAA)/CRISPR/Cas12a/lateral flow dipstick(LFD)/visual detection分类
农业科技引用本文复制引用
黄灿,吴浩雨,熊典广,黄华毅,田呈明..基于RAA-CRISPR/Cas12a-LFD的杜英疫病菌可视化检测技术的建立[J].北京林业大学学报,2025,47(4):10-20,11.基金项目
广东省林业科技创新项目(2020KJCX004、2023KJCX022). （2020KJCX004、2023KJCX022）
