灰葡萄孢RPA-CRISPR/Cas12a可视化检测方法的建立与应用

doi:10.3864/j.issn.0578-1752.2026.15.007

摘要/Abstract

摘要：

【目的】灰葡萄孢（Botrytis cinerea）是导致设施蔬菜灰霉病的重要病原菌，其寄主范围广泛、发病后传播速度快，易造成严重的经济损失。论文旨在建立灰霉病病害显症前的早期精准检测方法，为设施蔬菜灰霉病的早期监测与精准防控提供技术支持。【方法】以灰葡萄孢菌株B05.10的保守基因BC1G_05726为靶标，设计并筛选特异性重组酶聚合酶等温扩增（RPA）引物和CRISPR/Cas12a系统的crRNA。通过对反应时间、Cas12a与crRNA浓度比等关键参数进行系统优化，建立RPA-CRISPR/Cas12a快速可视化检测方法。利用便携式蓝光照射设备观察荧光信号，以评价检测体系的特异性、灵敏度及对实际样本（发病植株及环境空气样本）的检测效果。【结果】筛选获得最优crRNA（crRNA-1）及引物对（RPA-bc-F/R）。优化后的检测体系条件为RPA反应37 ℃扩增20 min，CRISPR/Cas12a反应中Cas12a与crRNA的终浓度分别为200和50 nmol·L^-1，反应20 min。全流程可在40 min内完成。灵敏度试验表明，该体系对灰葡萄孢基因组DNA的检测下限为10^-4 ng·μL^-1，较常规PCR灵敏度提高100倍。特异性检测结果显示，仅灰葡萄孢产生阳性荧光信号，其他14种供试真菌、细菌及阴性对照均无荧光，表明体系特异性良好。应用试验中，该方法可有效检测来自不同寄主的灰葡萄孢菌株。在人工接种黄瓜叶片后，侵染1 d即可稳定检出病原菌，早于症状出现时间。对设施环境空气样本的动态监测表明，接种后60 h即可检出环境中的灰葡萄孢，此时植株尚未显现典型病斑，验证了该方法的早期预警能力。【结论】建立的RPA-CRISPR/Cas12a可视化检测方法，可在恒温条件下快速、灵敏、特异地检测灰葡萄孢，且无需复杂仪器，结果肉眼可判读，适用于土壤、种子、植株及空气样本中病原菌的现场检测。

关键词: 灰葡萄孢, 重组酶聚合酶等温扩增, CRISPR/Cas12a检测体系, 可视化检测

Abstract:

【Objective】Botrytis cinerea, the causal agent of gray mold in facility vegetables, is characterized by a wide host range and rapid post-infection spread, often leading to severe economic losses. This paper aims to establish an early and precise detection method for gray mold before its visible symptoms appear, and to provide technical support for the early monitoring and targeted control of gray mold in facility vegetable cultivation.【Method】A conserved gene sequence (BC1G_05726) from B. cinerea strain B05.10 was used as the target. Specific recombinase polymerase isothermal amplification (RPA) primers and CRISPR/Cas12a crRNAs were designed and screened. Key parameters including reaction time and the concentration ratio of Cas12a to crRNA were systematically optimized to establish a rapid visual detection method combining RPA and CRISPR/Cas12a. Fluorescence signals were visualized using a portable blue light transilluminator. The specificity, sensitivity, and applicability of the developed method were evaluated using complex environmental samples (infested soil, seeds, symptomatic plants, and air samples).【Result】The optimal crRNA (crRNA-1) and primer pair (RPA-bc-F/R) were selected. The optimized detection conditions were as follows: RPA reaction at 37 ℃ for 20 min, followed by CRISPR/Cas12a reaction with final concentrations of 200 nmol·L^-1 for Cas12a and 50 nmol·L^-1 for crRNA for another 20 min. The entire detection process was completed within 40 min. The sensitivity assay revealed a detection limit of 10^-4 ng·μL^-1 of genomic DNA of B. cinerea, which was 100-fold more sensitive than conventional PCR. Specificity testing showed that only B. cinerea produced positive fluorescence signals, while none of the other 14 tested fungal and bacterial species or the negative control generated any signal, indicating excellent specificity of the assay. In application tests, the method successfully detected B. cinerea strains from different hosts. After artificial inoculation on cucumber leaves, the pathogen was stably detected as early as 1 d post-inoculation, preceding symptom appearance. Dynamic monitoring of air samples in the facility environment demonstrated that B. cinerea could be detected in the air at 60 h post-inoculation, at which time typical disease lesions had not yet developed on the plants, validating the early warning capability of this method.【Conclusion】The RPA-CRISPR/Cas12a-based visual detection method established in this study enables rapid, sensitive, and specific detection of B. cinerea under isothermal conditions without requiring complex instrumentation, and the results can be read by the naked eye. This method is suitable for on-site detection of the pathogen in soil, seed, plant, and air samples.

Key words: Botrytis cinerea, recombinase polymerase isothermal amplification (RPA), CRISPR/Cas12a detection system, visual detection

周雨欣, 申永俊, 朱广雪, 柴阿丽, 谢学文, 李磊, 范腾飞, 孙先花, 项盛, 李宝聚, 石延霞. 灰葡萄孢RPA-CRISPR/Cas12a可视化检测方法的建立与应用[J]. 中国农业科学, 2026, 59(15): 3328-3339.

ZHOU YuXin, SHEN YongJun, ZHU GuangXue, CHAI ALi, XIE XueWen, LI Lei, FAN TengFei, SUN XianHua, XIANG Sheng, LI BaoJu, SHI YanXia. Establishment and Application of RPA-CRISPR/Cas12a-based Visual Detection of Botrytis cinerea[J]. Scientia Agricultura Sinica, 2026, 59(15): 3328-3339.

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分类Taxon	病原菌Pathogen	菌株编号Strain ID	寄主Host	来源地Origin
真菌 Fungus	多主棒孢Corynespora cassiicola	HG14102524	黄瓜Cucumber	内蒙古赤峰Chifeng, Inner Mongolia
	尖孢镰孢Fusarium oxysporum	HG11082202	黄瓜Cucumber	北京Beijing
	茄匍柄霉Stemphylium solani	FQ2104160401	番茄Tomato	河南信阳Xinyang, Henan
	立枯丝核菌Rhizoctonia solani	HG09110801	黄瓜Cucumber	北京Beijing
	古巴假霜霉Pseudoperonospora cubensis	HG23103105	黄瓜Cucumber	北京Beijing
	核盘菌Sclerotinia sclerotiorum	HG11101715	黄瓜Cucumber	甘肃兰州Lanzhou, Gansu
	瓜果腐霉Pythium aphanidermatum	TG13082203	甜瓜Melon	河北沧州Cangzhou, Hebei
	葫芦科刺盘孢Colletotrichum orbiculare	HG09110501	黄瓜Cucumber	北京Beijing
	瓜枝孢霉Cladosporium cucumerinum	HG1603071610	黄瓜Cucumber	山东寿光Shouguang, Shandong
细菌 Bacillus	密执安棒形杆菌密执安亚种 Clavibacter michiganensis subsp. michiganensis	FQ20082701	番茄Tomato	北京Beijing
	丁香假单胞菌番茄致病变种 Pseudomonas syringae pv. tomato	FQ19051203	番茄Tomato	北京Beijing
	野油菜黄单胞Xanthomonas campestris	LJ1201090320	辣椒Chili	北京Beijing
	胡萝卜软腐果胶杆菌巴西亚种 Pectobacterium carotovorum subsp. brasiliense	DJ2008300205	豆角Green bean	北京Beijing
	茄科雷尔氏菌Ralstonia solanacearum	FQ21052304	番茄Tomato	北京Beijing

序列名称Sequence ID	序列Sequence (5′→3′)	功能Function
RPA-bc-F	CGACGACATCGAATTAGGTCCTGCTCCTC	介导等温扩增起始 Isothermal amplification initiation mediation
RPA-bc-R	CTTTCTCTGCCACTATTTTCTCGATATCAGTATG	介导等温扩增起始 Isothermal amplification initiation mediation
crRNA-1	UAAUUUCUACUAAGUGUAGAUCCCGAAGAAUCAGGACAGAGGCC	引导核酸酶靶向切割 Nuclease-guided targeted cleavage
crRNA-2	UAAUUUCUACUAAGUGUAGAUCAAGACAUGUAUUCAAAUUCA
crRNA-3	UAAUUUCUACUAAGUGUAGAUCCCGAAGAAUCAGGACAGAG
ssDNA-FQ	FAM-TTATT-BHQ1	报告靶标存在Target presence reporting

序号 No.	菌株编号 Strain number	寄主 Host	采集地点 Location	RPA-CRISPR/Cas12a检测结果 RPA-CRISPR/Cas12a result	PCR检测结果 PCR result
1	SC17032306	生菜Lettuce	河北唐山Tangshan, Hebei	+	+
2	QZ1501080206	茄子Eggplant	北京Beijing	+	+
3	XHL10041702	西葫芦Courgette	山东淄博Zibo, Shandong	+	+
4	QC1501070304	芹菜Celery	北京Beijing	+	+
5	FQ1501080425	番茄Tomato	北京Beijing	+	+
6	LJ1504147001	辣椒Chili	山东寿光Shouguang, Shandong	+	+
7	QK14121910	秋葵Okra	北京Beijing	+	+
8	CM16033104	草莓Strawberry	北京Beijing	+	+
9	KJ15011306	苦苣Chicory	北京Beijing	+	+
10	JC23021701	韭菜Garlic chives	北京Beijing	+	+
11	HG23021704	黄瓜Cucumber	北京Beijing	+	+