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

Abstract

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

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.

Figures/Tables 10

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Table 3

Fig. 5

Fig.6

Fig. 7

References 32

[1]	Rahman M U, Liu X, Wang X, Fan B. Grapevine gray mold disease: Infection, defense and management[J]. Horticulture Research, 2024, 11(9): uhae182.
[2]	Wang K, Li C, Lei C, Jiang Y, Qiu L, Zou X, Zheng Y. β-aminobutyric acid induces priming defence against Botrytis cinerea in grapefruit by reducing intercellular redox status that modifies posttranslation of VvNPR1 and its interaction with VvTGA1[J]. Plant Physiology and Biochemistry, 2020, 156: 552-565.
[3]	赵杨, 苗则彦, 李颖, 白元俊. 番茄灰霉病防治研究进展[J]. 中国植保导刊, 2014, 34(7): 21-29.
	Zhao Y, Miao Z Y, Li Y, Bai Y J. Research progress on controlling against tomato gray mold[J]. China Plant Protection, 2014, 34(7): 21-29.(in Chinese)
[4]	郑果, 杜蕙. 几种新型药剂对番茄灰霉病的防治效果[J]. 中国蔬菜, 2006(9): 22-23.
	Zheng G, Du H. Control effect of several new fungicides on tomato gray mold[J]. China Vegetables, 2006(9): 22-23.(in Chinese)
[5]	Liu S, Che Z, Chen G. Multiple-fungicide resistance to carbendazim, diethofencarb, procymidone, and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan Province, China[J]. Crop Protection, 2016, 84: 56-61.
[6]	Leroux P, Fritz R, Debieu D, Albertini C, Lanen C, Bach J, Gredt M, Chapeland F. Mechanisms of resistance to fungicides in field strains of Botrytis cinerea[J]. Pest Management Science, 2002, 58(9): 876-888.
[7]	Fernández-Ortuño D, Chen F, Schnabel G. Resistance to pyraclostrobin and boscalid in Botrytis cinerea isolates from strawberry fields in the carolinas[J]. Plant Disease, 2012, 96(8): 1198-1203.
[8]	Fan X, Zhang J, Yang L, Wu M, Chen W, Li G. Development of PCR-based assays for detecting and differentiating three species of Botrytis infecting broad bean[J]. Plant Disease, 2015, 99(5): 691-698.
[9]	Kennedy R, Wakeham A J, Byrne K G, Meyer U M, Dewey F M. A new method to monitor airborne inoculum of the fungal plant pathogens Mycosphaerella brassicicola and Botrytis cinerea[J]. Applied and Environmental Microbiology, 2000, 66(7): 2996-3003.
[10]	Ammour M S, Castaldo E, Fedele G, Rossi V. Use of LAMP for assessing Botrytis cinerea colonization of bunch trash and latent infection of berries in grapevines[J]. Plants, 2020, 9(11): 1538.
[11]	Celik M, Kalpulov T, Zutahy Y, Ish-Shalom S, Lurie S, Lichter A. Quantitative and qualitative analysis of Botrytis inoculated on table grapes by qPCR and antibodies[J]. Postharvest Biology and Technology, 2009, 52(2): 235-239.
[12]	Diguta C F, Rousseaux S, Weidmann S, Bretin N, Vincent B, Guilloux-Benatier M, Alexandre H. Development of a qPCR assay for specific quantification of Botrytis cinerea on grapes[J]. FEMS Microbiology Letters, 2010, 313(1): 81-87.
[13]	Boyle D S, McNerney R, Low H T, Leader B T, Pérez-Osorio A C, Meyer J C, O’Sullivan D M, Brooks D G, Piepenburg O, Forrest M S. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification[J]. PLoS ONE, 2014, 9(8): e103091.
[14]	Jiang Y, Qian F, Yang J, Liu Y, Dong F, Xu C, Sun B, Chen B, Xu X, Li Y, Wang R, Yang S. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum[J]. Nature Communications, 2017, 8(1): 15179.
[15]	Cheng Y, Tang X, Gao C, Li Z, Chen J, Guo L, Wang T, Xu J. Molecular diagnostics and pathogenesis of fungal pathogens on bast fiber crops[J]. Pathogens, 2020, 9(3): 223.
[16]	Gootenberg J S, Abudayyeh O O, Kellner M J, Joung J, Collins J J, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science, 2018, 360(6387): 439-444.
[17]	Liang X, Zhang X, Xi K, Liu Y, Jijakli M H, Guo W. Development of an RPA-based CRISPR/Cas12a assay in combination with a lateral flow strip for rapid detection of toxigenic Fusarium verticillioides in maize[J]. Food Control, 2024, 157: 110172.
[18]	Guo Y, Tan J, Jiao B, Wang F, Wang H, Yang C, Dai T. Establishment of a rapid detection system for Phytophthora syringae based on RPA/CRISPR-cas12a[J]. Crop Protection, 2025, 190: 107106.
[19]	Pahlich E, Gerlitz C. A rapid DNA isolation procedure for small quantities of fresh leaf tissue[J]. Phytochemistry, 1980, 19: 11-13.
[20]	赵芊, 李文, 李西柳, 贾振华, 封晓娟, 宋水山. 基于LAMP技术的番茄灰霉病早期快速检测[J]. 河南农业科学, 2025, 54(6): 84-91.
	Zhao Q, Li W, Li X L, Jia Z H, Feng X J, Song S S. Early and rapid detection of tomato gray mold based on LAMP technology[J]. Journal of Henan Agricultural Sciences, 2025, 54(6): 84-91.(in Chinese)
[21]	王莹莹, 柴阿丽, 孙阳, 李宝聚. 李宝聚博士诊病手记(九十二) 天津河北6种黄瓜叶部病害的症状诊断及防治建议[J]. 中国蔬菜, 2016(3): 78-80.
	Wang Y Y, Chai A L, Sun Y, Li Baoju’s diagnostic notes (XCII): Symptom diagnosis and control suggestions of six cucumber leaf diseases in Tianjin and Hebei[J]. China Vegetables, 2016(3): 78-80.(in Chinese)
[22]	张涛. 石河子地区葡萄烂果病的研究[D]. 石河子: 石河子大学, 2010.
	Zhang T. Study on grape rot disease in Shihezi area[D]. Shihezi: Shihezi University, 2010.(in Chinese)
[23]	Suarez M B, Walsh K, Boonham N, O’Neill T, Pearson S, Barker I. Development of real-time PCR (TaqMan^®) assays for the detection and quantification of Botrytis cinerea in planta[J]. Plant Physiology and Biochemistry, 2005, 43(9): 890-899.
[24]	汪少丽, 曲恒华, 王英姿, 王培松, 栾炳辉, 王冠华. 苹果轮纹病菌LAMP快速检测方法的建立[J]. 植物保护学报, 2020, 47(1): 127-133.
	Wang S L, Qu H H, Wang Y Z, Wang P S, Luan B H, Wang G H. Development of a loop-mediated isothermal amplification assay for rapid detection of apple ring rot pathogen Botryosphaeria dothidea[J]. Journal of Plant Protection, 2020, 47(1): 127-133.(in Chinese)
[25]	梁伊菲, 王春伟. 黄芪根腐病尖孢镰刀菌LAMP可视化快速检测方法的建立[J]. 山西农业科学, 2023, 51(4): 420-426.
	Liang Y F, Wang C W. Establishment of LAMP visual rapid detection method for Fusarium oxysporum of root rot of Astragalus membranaceus[J]. Journal of Shanxi Agricultural Sciences, 2023, 51(4): 420-426.(in Chinese)
[26]	Fan F, Wu M Y, Zhang H Q, Li G, Luo C. Rapid and simple detection of anilinopyrimidine resistance in Botrytis cinerea by combining recombinase polymerase amplification with the CRISPR/Cas12a assay[J]. Plant Disease, 2025, 109(9): 1831-1838.
[27]	Hussain R, Zhao B Y, Aarti A, Yin W X, Luo C X. A single tube RPA/Cas12a-based diagnostic assay for early, rapid, and efficient detection of Botrytis cinerea in sweet cherry[J]. Plant Disease, 2025, 109(6): 1244-1253.
[28]	Zhao W C, Lin M L, He Y L, Gao J, Zhang J L, Li M J, Yang Q X. Rapid visual field detection of Pratylenchus coffeae in yam tissues via RPA-CRISPR/Cas12a[J]. Crop Protection, 2025, 197: 107341.
[29]	Li W, Tang J, Ma Z, Zhang Y, Ye Z, Fu H. Development of an RPA-CRISPR/Cas12a-based rapid diagnosis strip for the tangerine pathotype of Alternaria alternata[J]. Microorganisms, 2025, 13(4): 826.
[30]	Anbazhagan P, Parameswari B, Anitha K, Chaitra G V, Bajaru B, Rajashree A, Mangrauthia S K, Yousuf F, Chalam V C, Singh G P. Advances in plant pathogen detection: Integrating recombinase polymerase amplification with CRISPR/Cas systems[J]. 3 Biotech, 2024, 14(9): 214.
[31]	Xu T, Zhang Y, Li S, Dai C, Wei H, Chen D, Zhao Y, Liu H, Li D, Chen P, Liu B F, Tian Y. Deep learning-enhanced hand-driven microfluidic chip for multiplexed nucleic acid detection based on RPA/CRISPR[J]. Advanced Science, 2025, 12(21): e2414918.
[32]	Giakoumoglou N, Kalogeropoulou E, Klaridopoulos C, Pechlivani E M, Christakakis P, Markellou E, Frangakis N, Tzovaras D. Early detection of Botrytis cinerea symptoms using deep learning multi- spectral image segmentation[J]. Smart Agricultural Technology, 2024, 8: 100481.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

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	+	+

Establishment and Application of RPA-CRISPR/Cas12a-based Visual Detection of Botrytis cinerea

RichHTML

PDF

Abstract

Cite this article

share this article

Figures/Tables 10

References 32

Related Articles 8

Metrics

Comments

Recommended 0

[1]	ZHANG DongMei, ZHOU XinXin, XIAO GuiLin, ZENG XiangGuo, WANG ChunYan, WANG ZeXian, HAN YongChao. Phenotypic Characteristics of Strawberry Floral Organs in Response to Botrytis cinerea Infection and Methods for Gray Mold Resistance Evaluation [J]. Scientia Agricultura Sinica, 2026, 59(7): 1456-1466.
[2]	ZHANG TianYu, LI Bai, ZANG JinPing, CAO HongZhe, DONG JinGao, XING JiHong, ZHANG Kang. Genome-Wide Identification and Expression Analysis of HMG Family Genes in Botrytis cinerea [J]. Scientia Agricultura Sinica, 2025, 58(4): 704-718.
[3]	ZHAO YuLei, XIN JiaLu, LI ChengNan, LI Shan, XIE XuFei, YIN Xiao. Screening of Target Genes Downstream of VviERF045, a Transcription Factor Associated with Gray Mold Resistance in Vitis vinifera [J]. Scientia Agricultura Sinica, 2025, 58(13): 2578-2590.
[4]	ZHANG XiaoQi, SHEN JianGuo, LIAO FuRong, LI WeiMin, JIN YuJie, WUFUER Shayidan, ZHENG LuPing. Establishment and Application of RT-RAA-CRISPR/Cas12a-Based Visual Detection of Prunus Necrotic Ringspot Virus [J]. Scientia Agricultura Sinica, 2025, 58(12): 2371-2381.
[5]	LI Jie, LIANG ZhiLin, SUN Yan, TAN GenJia, HUAI BaoYu. Functional Analysis of SlSnRK1.2 in Regulating Tomato Resistance to Grey Mould [J]. Scientia Agricultura Sinica, 2024, 57(21): 4238-4247.
[6]	SHAO ShuJun,HU ZhangJian,SHI Kai. The Role and Mechanism of Linoleyl Ethanolamide in Plant Resistance Against Botrytis cinerea in Tomato [J]. Scientia Agricultura Sinica, 2022, 55(9): 1781-1789.
[7]	GUO ZeXi,SUN DaYun,QU JunJie,PAN FengYing,LIU LuLu,YIN Ling. The Role of Chalcone Synthase Gene in Grape Resistance to Gray Mold and Downy Mildew [J]. Scientia Agricultura Sinica, 2022, 55(6): 1139-1148.
[8]	GONG ChangWei,QIN YiMan,QU JinSong,WANG XueGui. Resistance Detection and Mechanism of Strawberry Botrytis cinerea to Fludioxonil in Sichuan Province [J]. Scientia Agricultura Sinica, 2018, 51(22): 4277-4287.