柑橘衰退病毒RT-RPA-LFD可视化检测方法的建立及应用  

Establishment and application of RT-RPA-LFD visualization assay for rapid detection of citrus tristeza virus

作　　者：申世凯 曾婷 乔兴华 陈力 任杰群 周彦[1] SHEN Shikai;ZENG Ting;QIAO Xinghua;CHEN Li;REN Jiequn;ZHOU Yan(Southwest University,National Citrus Engineering Research Center,Chongqing 400712,China;Plant Protection and Fruit Technology Extension Station of Wanzhou District,Chongqing 400712,China;Chongqing Three Gorges Academy of Agricultural Sciences,Chongqing 400712,China)

机构地区：[1]西南大学柑桔研究所国家柑桔工程技术研究中心,重庆400712 [2]重庆市万州区植物保护与果树技术推广站,重庆400712 [3]重庆三峡农业科学院,重庆400712

出　　处：《果树学报》2023年第12期2652-2660,共9页Journal of Fruit Science

基　　金：财政部和农业农村部国家现代农业产业技术体系(CARS-26-05B)。

摘　　要：【目的】柑橘衰退病由柑橘衰退病毒(citrus tristeza virus,CTV)引起,是一种世界性的重要柑橘病害。为实现CTV的田间快速检测,建立一种准确、快速且可视化的检测方法。【方法】以CTV外壳蛋白(CP)的保守区域为靶标,设计3对特异性引物和探针,通过引物筛选,以及优化引物浓度、反应时间和反应温度等条件,建立CTV的反转录-重组酶聚合酶扩增-侧流层析试纸条(RT-RPA-LFD)快速检测方法,明确其灵敏度,并用于田间疑似样品的检测。【结果】建立了CTV的RT-RPA-LFD检测方法:最佳检测引物为RPA-1F/R,对应探针为RPA-P,最佳反应条件为40℃,25 min,且与其他5种柑橘病毒无交叉反应。该方法的灵敏度是RT-PCR的100倍,最低可检测到2.12×10^(1)拷贝·μL^(-1)的CTV核酸,与RT-qPCR相当。采用RT-RPA-LFD法在67份田间样品中检测出CTV阳性样品41份,与RT-PCR法检测结果一致。【结论】建立的CTV RT-RPA-LFD法具有操作简单、快速、结果可视等优点,适合基层植保工作者对田间样品开展快速检测。【Objective】Tristeza caused by citrus tristeza virus(CTV)is one of the most destructive cit-rus diseases in the world,which is mainly spread by several aphid species and bud-grafting.Severe CTV isolates could cause quick decline of sour orange rootstock,and stem pitting of susceptible culti-vars.In recent years,stunted,severe stem pitting and reduced fruit quality were observed in Newhall na-vel orange and some tangor cultivars,causing severe economic losses in major citrus-growing provinc-es of China,especially in Hunan,Jiangxi,Yunnan,Sichuan provinces.Prompt and accurate CTV detec-tion in the nursery and field samples is necessary to control CTV.To date,serological techniques,re-verse transcription PCR(RT-PCR),RT-real-time PCR(RT-qPCR)and other methods have been used to detect CTV.However,these traditional detection techniques are generally flawed.The purpose of this study was to establish a reliable,accurate,convenient and visual reverse transcription-recombinase poly-merase amplification(RT-RPA)combined with lateral flow dipstick(LFD)method for CTV detection.【Methods】Three pairs of primers and a specific probe used for CTV detection were designed accord-ing to the conservative sequence of the coat protein(CP)gene of CTV isolates(NCBI number MH558665.1,MH558666.1,JX266712.1,JQ911664.1 and JQ061137.1)from China.By detecting CTV-infected citrus samples,primers with the best specificity and amplification efficiency were selected to establish the RT-RPA-LFD for CTV detection.The total RNAs were extracted from 100 mg CTV-infect-ed citrus leaf samples using RNAiso Plus and used for CTV detection.The reverse transcription was performed using a C1000 Thermal Cycler in a 20μL reaction mix containing 1μL of Oligo dT Primer,1μL of 10μmol·L^(-1)dNTP Mixture,1μL of RNA template,4μL of PrimeScript Buffer,0.5μL of RNase Inhibitor,and 1μL of PrimeScript RTase.The reaction was carried out for 45 min at 42℃and 5 min at 95℃.RT-RPA-LFD reaction system was optimized with respect to the primer concentration(1,2.

关 键 词：柑橘 柑橘衰退病毒 反转录-重组酶聚合酶扩增-侧流层析试纸条 快速检测 

分 类 号：S666[农业科学—果树学]