假禾谷镰孢RPA-CRISPR/Cas12a快速检测体系的建立与应用

Development and application of rapid detection system for Fusarium pseudograminearum based on RPA-CRISPR/Cas12a

假禾谷镰孢菌Fusarium pseudograminearum是引起我国黄淮麦区小麦茎基腐病的优势病原菌,对其精准、快速的检测有助于小麦茎基腐病的监测预警和精准防控。本研究基于F.pseudograminearum的NPS15基因,建立了重组酶聚合酶等温扩增技术(RPA)结合CRISPR/Cas12a的快速检测体系,该体系在37℃恒温条件下,40 min内可完成对目标病原菌的检测,检测灵敏度可达10^(–3)pg/μL,检测结果可通过核酸试纸条颜色反应直观读取,具有特异、灵敏、快速等优点。经验证,该体系可从带菌土壤、种子以及发病植株等复杂样本中检测到目标病原菌,为在田间实时快速检测F.pseudograminearum提供了一种高效方法。

Fusarium pseudograminearum is the dominant pathogen causing wheat crown rot in the Huanghuai wheat-growing region in China.Accurate and rapid detection techniques are beneficial for monitoring and early warning of wheat crown rot.In this study,a rapid detection system combining RPA(recombinase polymerase amplification)with CRISPR/Cas12a was established based on the NPS15 gene of F.pseudograminearum,which can complete the detection of the target pathogen in 40 min at a constant temperature of 37℃,with a sensitivity of 10^(–3)pg/μL.The test results can be visually interpreted through the color reaction of nucleic acid test strips,with advantages of specificity,sensitivity,and rapidity.The system was validated to be able to detect the target pathogen from complex samples such as soil,seeds,and infected plants,providing an efficient method for the rapid detection of F.pseudograminearum in the field.