李属坏死环斑病毒RT-RAA-CRISPR/Cas12a可视化检测方法的建立与应用

doi:10.3864/j.issn.0578-1752.2025.12.008

中国农业科学 ›› 2025, Vol. 58 ›› Issue (12): 2371-2381.doi: 10.3864/j.issn.0578-1752.2025.12.008

李属坏死环斑病毒RT-RAA-CRISPR/Cas12a可视化检测方法的建立与应用

张晓琪¹(), 沈建国²(), 廖富荣³, 李为民⁴, 金雨洁¹, 沙依旦·吾甫尔¹, 郑璐平¹()

¹ 福建农林大学植物病毒研究所，福州 350002
² 福州海关技术中心，福州 350001
³ 厦门海关技术中心，福建厦门 361026
⁴ 北京农学院植物保护系/农业农村部华北都市农业重点实验室，北京 102206

收稿日期:2025-03-10 接受日期:2025-04-29 出版日期:2025-06-19 发布日期:2025-06-19
通信作者:
郑璐平，E-mail：lupingz@126.com。
沈建国，E-mail：shenjg_agri@163.com
联系方式: 张晓琪，E-mail:zhangxiaoqi202202@163.com
基金资助:
福建省自然科学基金面上项目(2023J01441); 福建省检验检疫技术研究重点实验室开放课题(FJKF2023-02); 福建农林大学科技创新专项基金(KFB23029)

Establishment and Application of RT-RAA-CRISPR/Cas12a-Based Visual Detection of Prunus Necrotic Ringspot Virus

ZHANG XiaoQi¹(), SHEN JianGuo²(), LIAO FuRong³, LI WeiMin⁴, JIN YuJie¹, WUFUER Shayidan¹, ZHENG LuPing¹()

¹ Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002
² Technology Center of Fuzhou Custom District, Fuzhou 350001
³ Xiamen Customs District Technology Center, Xiamen 361026, Fujian
⁴ Department of Plant Protection, Beijing University of Agriculture/Key Laboratory for Northern Urban Agriculture, Ministry of Agriculture and Rural Affairs, Beijing 102206

Received:2025-03-10 Accepted:2025-04-29 Published:2025-06-19 Online:2025-06-19

摘要/Abstract

摘要：

【目的】利用逆转录重组酶介导的核酸等温扩增技术（reverse transcriptase recombinase-aided amplification，RT-RAA）结合CRISPR/Cas12a系统，建立李属坏死环斑病毒（prunus necrotic ringspot virus，PNRSV）的RT-RAA-CRISPR/ Cas12a可视化检测方法。【方法】依据PNRSV外壳蛋白（coat protein，CP）编码基因的保守序列，设计并筛选扩增效率高、特异性强的引物；针对引物和探针的浓度、扩增体系、反应温度和时间等条件进行优化，建立PNRSV的RT-RAA-CRISPR/Cas12a可视化检测方法。利用该方法对PNRSV、李痘病毒（plum pox virus，PPV）、苹果花叶病毒（apple mosaic virus，ApMV）、黄瓜花叶病毒（cucumber mosaic virus，CMV）、马铃薯X病毒（potato virus X，PVX）、马铃薯Y病毒（potato virus Y，PVY）等李属植物常见病毒进行检测，验证方法的特异性；将PNRSV的总RNA进行10倍梯度稀释，分别采用RT-PCR、RT-RAA以及RT-RAA-CRISPR/Cas12a方法进行检测，比较3种方法的灵敏度；对口岸收集的31份疑似感染病毒的桃果实试验样品进行RT-RAA-CRISPR/Cas12a和RT-PCR方法检测，验证该可视化检测方法的实用性。【结果】成功建立了用于检测PNRSV的RT-RAA-CRISPR/Cas12a可视化检测方法。最终体系优化结果为引物RT-RAA-PNRSV-F2/R2、荧光报告基因FQ、CRISPR-Cas12a、PNRSV-crRNA（CRISPR RNA，crRNA）的工作浓度分别为0.4 μmol·L^-1、800、200、240 nmol·L^-1，反应条件为41 ℃下反应45 min。该方法可特异性检测PNRSV，与李属植物常见病毒无交叉反应。对携带PNRSV的桃果实样品RNA的检测灵敏度，RT-RAA和RT-RAA-CRISPR/Cas12a分别可达3.06 pg·μL^-1和306 fg·μL^-1，RT-RAA-CRISPR/Cas12a灵敏度是RT-RAA和RT-PCR的10倍。口岸收集的31份桃果实试验样品中，RT-PCR检出14份阳性样品，RT-RAA-CRISPR/Cas12a检出15份阳性样品，两者检测结果一致率基本一致。【结论】建立了PNRSV的RT-RAA-CRISPR/Cas12a可视化检测方法，该方法具有简便、快速、高灵敏度、高特异性和直观的特点，适用于现场快速检测PNRSV。

关键词: 李属坏死环斑病毒, RT-RAA, CRISPR/Cas12a, 可视化检测

Abstract:

【Objective】The study aims to establish a novel visual detection technique for prunus necrotic ringspot virus (PNRSV) by combining reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR/Cas12a system (RT-RAA-CRISPR/ Cas12a).【Method】Primers with high amplification efficiency and strong specificity were designed and selected based on the conserved regions of the coat protein (CP) gene of PNRSV. The detection conditions, including primer, probe concentration, temperature, and reaction time were optimized to develop a visual detection method for PNRSV by RT-RAA-CRISPR/Cas12a technology. The specificity of this method was evaluated by detecting PNRSV and common Prunus viruses, including plum pox virus (PPV), apple mosaic virus (ApMV), cucumber mosaic virus (CMV), potato virus X (PVX), and potato virus Y (PVY). The total RNAs from PNRSV-infected fruit were diluted in 10-fold gradients, then RT-PCR, RT-RAA and RT-RAA-CRISPR/Cas12a were performed to compare the sensitivity of the three methods. The RT-RAA-CRISPR/Cas12a and RT-PCR methods were used to detect 31 peach fruit test samples suspected to be infected with the virus collected at the port to verify the practicability of the visual detection method.【Result】The RT-RAA-CRISPR/Cas12a-based visual detection method for PNRSV was successfully established. The optimized working concentrations were as follows: RT-RAA-PNRSV-F2/R2 primers at 0.4 μmol·L^-1, fluorescent reporter (FQ) at 800 nmol·L^-1, CRISPR-Cas12a at 200 nmol·L^-1, and PNRSV-crRNA (CRISPR RNA) at 240 nmol·L^-1, the reaction conditions were performed at 41 ℃ for 45 min. This method showed high specificity for PNRSV and had no cross-reaction with other common Prunus viruses. The limit of detection for PNRSV RNA in peach fruit samples reached 3.06 pg·μL^-1 and 306 fg·μL^-1 using RT-RAA and RT-RAA-CRISPR/Cas12a methods, respectively, showing the sensitivity of RT-RAA-CRISPR/Cas12a was 10 times higher than that of RT-RAA and RT-PCR. Among the 31 tested peach fruit samples at the port, 14 positive samples were identified by RT-PCR, while 15 positive samples were found by RT-RAA-CRISPR/Cas12a, indicating a high level of consistency between the two methods.【Conclusion】The RT-RAA-CRISPR/Cas12a visual detection method for PNRSV has been established. It is characterized by simplicity, rapidity, high sensitivity, high specificity, and visual readability, making it well-suited for rapid on-site detection of PNRSV.

Key words: prunus necrotic ringspot virus (PNRSV), RT-RAA, CRISPR/Cas12a, visual detection

张晓琪, 沈建国, 廖富荣, 李为民, 金雨洁, 沙依旦·吾甫尔, 郑璐平. 李属坏死环斑病毒RT-RAA-CRISPR/Cas12a可视化检测方法的建立与应用[J]. 中国农业科学, 2025, 58(12): 2371-2381.

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.

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方法Method	引物名称Primer name	引物序列Primer sequence (5′ to 3′)	靶标长度Target size (bp)
RT-PCR	RT-PCR-PNRSV-F	TAGGGTTTCGAGCGGTATAGGA	525
RT-PCR	RT-PCR-PNRSV-R	TCATCGACCAGCAAGACATCAG	525
RT-RAA	RT-RAA-PNRSV-F1	GAATAACCCGAATAGGAATAGGAACCCGAATA	220
	RT-RAA-PNRSV-R1	CAACTGAGGGAGAGTGGTCGTGAAGTCAATAC	220
	RT-RAA-PNRSV-F2	CGAATAACCCGAATAGGAATAGGAACCCGA	310
	RT-RAA-PNRSV-R2	TTATAGTCCTCCACCATCCCAATCCAACCATT	310
	RT-RAA-PNRSV-F3	TATTGACTTCACGACCACTCTCCCTCAGTTGA	320
	RT-RAA-PNRSV-R3	CACTTACCACTACGTACAAATCCCTAACCAA	320
	RT-RAA-PNRSV-F4	GAGGTGACGACGACAGAGGCAGTGAAGTAC	352
	RT-RAA-PNRSV-R4	TTACCACTACGTACAAATCCCTAACCAAGACC	352

李属坏死环斑病毒RT-RAA-CRISPR/Cas12a可视化检测方法的建立与应用

Establishment and Application of RT-RAA-CRISPR/Cas12a-Based Visual Detection of Prunus Necrotic Ringspot Virus

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试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)	试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)
1	400	480	9	400	240
2	300	480	10	300	240
3	200	480	11	200	240
4	100	480	12	100	240
5	400	360	13	400	120
6	300	360	14	300	120
7	200	360	15	200	120
8	100	360	16	100	120

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[2]	韩丽娟,刘文洪. 侵染百合的李属坏死环斑病毒病（PNRSV）的研究[J]. 中国农业科学, 2007, 40(9): 1943-1951 .

试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)	试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)
1	400	480	9	400	240
2	300	480	10	300	240
3	200	480	11	200	240
4	100	480	12	100	240
5	400	360	13	400	120
6	300	360	14	300	120
7	200	360	15	200	120
8	100	360	16	100	120

试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)	试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)
1	400	480	9	400	240
2	300	480	10	300	240
3	200	480	11	200	240
4	100	480	12	100	240
5	400	360	13	400	120
6	300	360	14	300	120
7	200	360	15	200	120
8	100	360	16	100	120