[1]侯雨萌,赵振兴,王思元,等.基于RT-RAA和CRISPR/Cas12a的一管式玉米矮花叶病毒快速检测方法[J].江苏农业学报,2025,(02):268-275.[doi:doi:10.3969/j.issn.1000-4440.2025.02.007]
　HOU Yumeng,ZHAO Zhenxing,WANG Siyuan,et al.A one-tube rapid detection method for maize dwarf mosaic virus based on RT-RAA and CRISPR/Cas12a[J].,2025,(02):268-275.[doi:doi:10.3969/j.issn.1000-4440.2025.02.007]
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基于RT-RAA和CRISPR/Cas12a的一管式玉米矮花叶病毒快速检测方法()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2025年02期
页码:
268-275
栏目:
植物保护
出版日期:
2025-02-28

文章信息/Info

Title:
A one-tube rapid detection method for maize dwarf mosaic virus based on RT-RAA and CRISPR/Cas12a
作者:
侯雨萌1; 2; 赵振兴1; 王思元1; 董铮1; 胡中泽3; 周涛2; 张永江1
(1. 中国检验检疫科学研究院,北京100176；2.中国农业大学植物保护学院,北京100193；3.江苏省农业科学院泰州农业科学研究所,江苏泰州225300)
Author(s):
HOU Yumeng1; 2; ZHAO Zhenxing1; WANG Siyuan1; DONG Zheng1; HU Zhongze3; ZHOU Tao2; ZHANG Yongjiang1
(1.Chinese Academy of Inspection and Quarantine, Beijing 100176, China；2.College of Plant Protection, China Agricultural University, Beijing 100193, China；3.Taizhou Institute of Agricultural Sciences, Jiangsu Academy of Agricultural Sciences, Taizhou 225300, China)
关键词:
玉米矮花叶病毒; 反转录重组酶介导等温核酸扩增（RT-RAA）技术; CRISPR/Cas12a; 侧向流动试纸条
Keywords:
maize dwarf mosaic virus;  reverse transcription-recombinase aided amplification (RT-RAA) technology; CRISPR/Cas12a; lateral flow dipstick
分类号:
S513
DOI:
doi:10.3969/j.issn.1000-4440.2025.02.007
文献标志码:
A
摘要:
玉米矮花叶病毒(Maize dwarf mosaic virus,MDMV)是一种重要的检疫性病毒,严重影响玉米的生产。为了有效防治玉米矮花叶病,本研究基于反转录重组酶介导等温核酸扩增(RT-RAA)技术和CRISPR/Cas12a系统构建了一种MDMV快速检测方法,将所有试剂集中在一个试管中进行反应,并通过侧向流动试纸条(LFD)检测反应结果。本研究筛选得到的最佳反应条件为,引物添加量2.8 μL、FB报告分子浓度100 nmol/L、RT-RAA系统反应时间20 min、CRISPR/Cas12a系统反应时间20 min。本研究构建的基于RT-RAA和CRISPR/Cas12a的一管式玉米矮花叶病毒快速检测方法特异性强、灵敏度高,且所用试验材料方便携带和运输,适用于现场检测。
Abstract:
Maize dwarf mosaic virus (MDMV) is an important quarantine virus that seriously affects maize production. To effectively prevent and control maize dwarf mosaic disease, this study constructed a rapid detection method for MDMV based on reverse transcription-recombinase aided amplification (RT-RAA) technology and the CRISPR/Cas12a system. All reagents were reacted in a single test tube, and the reaction results were detected by lateral flow dipstick (LFD). The optimal reaction conditions screened in this study were as follows: primer addition amount of 2.8 μL, FB reporter molecule concentration of 100 mol/L, RT-RAA system reaction time of 20 minutes, and CRISPR/Cas12a system reaction time of 20 minutes. The one-tube rapid detection method for maize dwarf mosaic virus based on RT-RAA and CRISPR/Cas12a constructed in this study has strong specificity, high sensitivity, and the experimental materials used are convenient for carrying and transportation, making it suitable for on-site detection.

参考文献/References:

[1]马丽. 玉米矮花叶病毒基因遗传转化研究[D]. 泰安:山东农业大学,2007.
[2]BEVERS N, OHLSON E W, KC K, et al. Sugarcane mosaic virus detection in maize using UAS multispectral imagery[J]. Remote Sensing,2024,16(17):3296-3296.
[3]DULANJANI W, AKHTAR A. Complete Genome characterization and coat protein genealogy of isolates of maize dwarf mosaic virus from johnsongrass and maize in Oklahoma and Missouri[J]. Plant Disease,2020,104(4):1214-1223.
[4]KUSIA E S, SUBRAMANIAN S, NYASANI J O, et al. First report of lethal necrosis disease associated with co-infection of finger millet with maize chlorotic mottle virus and sugarcane mosaic virus in Kenya[J]. Plant Disease,2015,99(6):899.
[5]田逸英,亓芳芳,焦志远,等. 四种中国检疫性玉米病毒的研究进展[J]. 植物保护学报,2022,49(5):1299-1312.
[6]ACHON M A, ALONSO-DUENAS N, SERRANO L. Maize dwarf mosaic virus diversity in the Johnsongrass native reservoir and in maize:evidence of geographical,host and temporal differentiation[J]. Plant Pathology,2011,60(2):369-377.
[7]陈红运,陈青,方志鹏,等. 进口甜玉米种子中玉米矮花叶病毒的检测[J]. 植物检疫,2023,37(4):62-65.
[8]赵振兴,范奇璇,冯黎霞,等. 基于重组酶聚合酶扩增技术的玉米矮花叶病毒快速检测方法的建立[J]. 植物保护,2022,48(6):83-89.
[9]王海光. 玉米矮花叶病流行原因分析及预测方法[D]. 北京:中国农业大学,2003.
[10]周伦理. 玉米矮花叶病研究进展[J]. 基因组学与应用生物学,2010,29(2):396-401.
[11]DULANJANI W, AKHTAR A. Evolutionary study of maize dwarf mosaic virus using nearly complete genome sequences acquired by next-generation sequencing[J]. Scientific Reports,2021,11(1):18786.
[12]LUO L L, CHANG Q R, WANG Q, et al. Identification and severity monitoring of maize dwarf mosaic virus infection based on hyperspectral measurements[J]. Remote Sensing,2021,13(22):4560.
[13]ZHANG Z Y, WANG Y G, SHEN X J, et al. RNA interference-mediated resistance to maize dwarf mosaic virus [J]. Plant Cell,Tissue and Organ Culture,2013,113(3):571-578.
[14]TRZMIEL K, JEZEWSKA M. Identification of maize dwarf mosaic virus in maize in Poland[J]. Plant Disease,2008,92(6):981.
[15]刘静远,于翠,黄昱萌,等. 基于MaxEnt模型预测玉米矮花叶病毒的潜在适生区[J]. 植物保护学报,2022,49(5):1383-1391.
[16]朱崧琪,梁柱伟,汪绍文. 《中华人民共和国进境植物检疫性有害生物名录》更新项目简介[J]. 中国海关,2021(7):54.
[17]FAN Z F, CHEN H Y, LIANG X M, et al. Complete sequence of the genomic RNA of the prevalent strain of a potyvirus infecting maize in China[J]. Archives of Virology,2003,148(4):773-782.
[18]LU X Y, XU H, SONG W, et al. Rapid and simple detection of Phytophthora cactorum in strawberry using a coupled recombinase polymerase amplification-lateral flow strip assay[J]. Phytopathology Research,2021,3(1):12.
[19]DU X J, ZANG Y, LIU H, et al. Rapid detection of staphylococcus aureus via recombinase polymerase amplification combined with lateral flow strip[J]. Food Analytical Methods,2018,11(8):2296-2306.
[20]凌莉,席静,王莹,等. 重组酶聚合酶扩增技术(RPA)检测创伤弧菌[J]. 江苏农业科学,2020,48(4):73-76.
[21]陈海云,滕丽琼,邱英华,等. 柑橘黄龙病RAA-LFD检测方法的建立[J]. 现代农业科技,2023(1):61-66.
[22]张永江,魏霜,袁俊杰,等. 一步法逆转录重组酶聚合酶常温扩增(RT-RPA)技术检测菜豆荚斑驳病毒[J]. 江苏农业科学,2018,46(21):96-98.
[23]单长林,周圆,任琰,等. 玉米内州萎蔫病菌荧光重组酶介导等温扩增检测方法的建立[J]. 浙江农业学报,2021,33(9):1676-1685.
[24]PAUL B, MONTOYA G. CRISPR-Cas12a:functional overview and applications[J]. Biomedical Journal,2020,43(1):8-17.
[25]路子显. 近六十年我国玉米产业发展、贸易变化与未来展望[J]. 黑龙江粮食,2021(9):9-14.
[26]LI Y D, WANG X F, XU R, et al. Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B[J]. BMC Microbiology,2023,23(1):333.
[27]朱裕敏,吴江,廖立珊,等. 鲤春病毒血症病毒RT-RAA-LFD检测方法的建立[J]. 中国预防兽医学报,2023,45(9):916-921.
[28]蒋军喜. 玉米矮花叶病病原鉴定、检测及外壳蛋白基因变异研究[D]. 杭州:浙江大学,2002.
[29]DA N T, SU L H, THANH H T, et al. Design an RPA model for the fulfillment process in an SAP ERP system[J]. Journal of the Chinese Institute of Engineers,2023,46(6):683-691.
[30]WIJAYASEKARA D, ALI A. First report of maize dwarf mosaic virus in johnsongrass in Oklahoma[J]. Plant Disease,2017,101(5):850.

备注/Memo

备注/Memo:
收稿日期:2024-07-18基金项目:国家重点研发计划项目(2021YFD1400100、2021YFD1400103)作者简介:侯雨萌(2000-),女,天津人,硕士研究生,主要从事玉米矮花叶病毒的研究；(E-mail)2192494895@qq.com。赵振兴为共同第一作者。通讯作者:张永江,(E-mail)zhangyjpvi@yeah.net；周涛,(E-mail)taozhousig@163.com
更新日期/Last Update: 2025-03-27