Establishment of RT‑RAA‑CRISPR/Cas12a Based Visual Detection Method for Pepper Mild Mottle Virus

doi:10.15933/j.cnki.1004-3268.2024.09.008

Abstract

Abstract: Pepper is an important horticultural crop.Pepper mild mottle virus(PMMoV)threatens pepper and other solanaceous crops’production.In order to improve the prevention and control efficiency of PMMoV,this study designed specific RAA primers based on the conserved sequence of its gene encoding coat protein(CP) to achieve rapid isothermal amplification of PMMoV based on recombinase‑aided amplification(RAA).And crRNA was designed to target the RT‑RAA amplification products based on the CRISPR/Cas12a system.The RT‑RAA‑CRISPR/Cas12a system was optimized,and the results showed that the strongest signal was observed in the total system at a final concentration of 400 nmol/L for reporter gene FQ,and a ratio of 1∶5 for Cas12a∶crRNA at the final concentrations of 200 nmol/L and 1 000 nmol/L.The final RT‑RAA amplification reaction and CRISPR chromogenic reaction each only took 15 min,and the positive signal could be directly observed under portable blue light irradiation equipment.This method could specifically detect PMMoV,and the detection limit of RNA of pepper samples carrying PMMoV could reach 1.34 pg/μL,which was 1 000 and 10 times more sensitive than that of conventional RT‑PCR and real‑time RT‑PCR.The detection results of 30 samples showed that the RT‑RAA‑CRISPR/Cas12a assay established in this study could detect PMMoV in leaves,fruits and soils of infected pepper and tomato plants.The established detection system could be used for rapid and sensitive visual detection of pepper mild mottle virus.

Key words: Pepper mild mottle virus, RT?RAA, CRISPR/Cas12a, Visual detection

摘要： 辣椒是重要的园艺作物，辣椒轻斑驳病毒（Pepper mild mottle virus，PMMoV）严重威胁辣椒等茄科园艺作物的生产安全。为提高PMMoV的防控效率，根据其编码外壳蛋白（Coat protein，CP）的基因保守序列，基于重组酶介导等温核酸扩增技术（Recombinase‑aided amplification，RAA），设计了特异性RAA引物，实现对PMMoV的快速等温扩增，并基于CRISPR/Cas12a的设计原则，设计了crRNA靶向RT-RAA扩增产物。优化结果显示，总反应体系在报告基因FQ终浓度为400 nmol/L，Cas12a/crRNA比例为1∶5、终浓度为200 nmol/L和1 000 nmol/L条件下检测信号最强，最终的RT-RAA反应和CRISPR显色体系分别仅需15 min，即可在便携式蓝光照射设备下直接观察到阳性信号。该方法可特异性检测PMMoV，对携带PMMoV的辣椒样品RNA的检测极限可达到1.34 pg/μL，分别是普通RT-PCR和实时荧光RT-PCR检测灵敏度的1 000倍和10倍。对实际样品中的检测结果显示，建立的RT-RAA-CRISPR/Cas12a检测技术可以在PMMoV侵染的辣椒和番茄叶片、果实及土壤中检测到PMMoV，可用于辣椒轻斑驳病毒的快速、灵敏、可视化检测。

关键词: 辣椒轻斑驳病毒, RT-RAA, CRISPR/Cas12a, 可视化检测

CLC Number:

S432.4⁺1

ZHAO Zhenxing, FAN Qixuan, WANG Siyuan, DONG Zheng, HU Zhongze, ZHANG Yongjiang. Establishment of RT‑RAA‑CRISPR/Cas12a Based Visual Detection Method for Pepper Mild Mottle Virus[J]. Journal of Henan Agricultural Sciences, 2024, 53(9): 80-87.

赵振兴, 范奇璇, 王思元, 董铮, 胡中泽, 张永江. 辣椒轻斑驳病毒RT-RAA-CRISPR/Cas12a可视化检测方法的建立[J]. 河南农业科学, 2024, 53(9): 80-87.

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