甘薯褪绿矮化病毒西非株系RT-RPA-LFD检测方法的建立与应用  

Establishment and Application of RT-RPA-LFD Detection Method for Sweet Potato Chlorotic Stunt Virus WA Strain

作　　者：王永江[1] 乔奇[1] 王爽[1] 赵付枚 田雨婷[1] 张德胜[1] 张振臣[1] WANG YongJiang;QIAO Qi;WANG Shuang;ZHAO FuMei;TIAN YuTing;ZHANG DeSheng;ZHANG ZhenChen(Institute of Plant Protection,Henan Academy of Agricultural Sciences/Henan Key Laboratory of Crop Pest Control/IPM Key Laboratory in Southern Part of North China,Ministry of Agriculture and Rural Affairs,Zhengzhou 450002)

机构地区：[1]河南省农业科学院植物保护研究所/河南省农作物病虫害防治重点实验室/农业农村部华北南部作物有害生物综合治理重点实验室,郑州450002

出　　处：《中国农业科学》2024年第14期2781-2790,共10页Scientia Agricultura Sinica

基　　金：国家甘薯产业技术体系(CARS-10-GW15);河南省科技攻关项目(232102111098);河南省农业科学院自主创新项目(2023ZC047,2024ZC058)。

摘　　要：【目的】利用逆转录酶重组酶聚合酶扩增(reverse transcriptase recombinase polymerase amplification,RT-RPA)结合侧向流层析(lateral flow dipstick,LFD)试纸条技术,建立甘薯褪绿矮化病毒(sweet potato chlorotic stunt virus,SPCSV)西非株系(west African strain,WA)的RT-RPA-LFD检测方法。【方法】根据SPCSV-WA外壳蛋白基因和热激蛋白基因的保守序列设计并筛选扩增效果好、特异性强的引物和探针。然后对引物和探针的浓度、扩增体系、温度、反应时间等条件进行优化,建立SPCSV-WA的RT-RPA-LFD检测方法。利用该方法对SPCSV-EA、甘薯羽状斑驳病毒(sweet potato feathery mottle virus,SPFMV)、甘薯潜隐病毒(sweet potato latent virus,SPLV)、甘薯G病毒(sweet potato virus G,SPVG)等常见的甘薯病毒进行检测,验证方法的特异性;将感染SPCSV-WA甘薯叶片样品总RNA进行10倍梯度稀释,分别采用RT-PCR和RT-RPA-LFD进行检测,比较RT-PCR和RT-RPA-LFD方法的灵敏度;对采自田间的甘薯样品和试管苗样品进行RT-RPA、RT-RPA-LFD和RT-PCR检测,验证该方法的实用性。【结果】建立了SPCSV-WA的RT-RPA-LFD检测方法,最适引物为CSV357F/R,探针CSV-CP-Probe(47 bp),引物和探针工作浓度分别为0.2和0.06μmol·L^(-1),温度为42℃,时间5 min。该方法可对SPCSV-WA进行特异性检测,与甘薯上其他常见病毒无交叉反应。RT-RPA-LFD最低可检测到总RNA稀释至10^(-4)溶液,而RT-PCR最低检测到总RNA稀释至10^(-3)溶液,RT-RPA-LFD灵敏度是RT-PCR的10倍。田间采集的22份甘薯样品经RT-PCR、RT-RPA和RT-RPA-LFD检测,均检出11份阳性样品,3种方法检测结果一致。28份试管苗样品的检测结果表明,RT-PCR和RT-RPA-LFD检测结果一致,均检出5份阳性样品。【结论】建立了SPCSV-WA的RT-RPA-LFD检测方法,该方法具有快速、简便、特异、灵敏、可视的特点,既可用于甘薯脱毒试管苗样品的病毒检测,也适用于基层单位田间甘薯病毒样品的现场快速检测�【Objective】This study aims to establish a novel technique for detecting the west African strain of sweet potato chlorotic stunt virus(SPCSV-WA)by combining reverse transcriptase recombinase polymerase amplification and lateral flow dipstick(RT-RPA-LFD).【Method】Primers and probes with good amplification results and strong specificity were designed and screened on the basis of the conserved sequences of the SPCSV-WA coat protein gene and heat shock protein gene.Then,the conditions such as primer and probe concentrations,amplification system,temperature,and reaction time were optimized to detect SPCSV-WA using RT-RPA-LFD.This method was used to detect common viruses on sweet potato such as the east African strain of sweet potato chlorotic stunt virus(SPCSV-EA),sweet potato feathery mottle virus(SPFMV),sweet potato latent virus(SPLV)and sweet potato virus G(SPVG)to verify the specificity.Total RNA from SPCSV-infected sweet potato leaves was diluted in a 10-fold gradient,and RT-PCR and RT-RPA-LFD were performed to compare the sensitivity of the two methods.Field sweet potato samples and test tube seedling samples were subjected to RT-RPA,RT-RPA-LFD,and RT-PCR detection to validate the practicality of the method.【Result】The RT-RPA-LFD detection method for SPCSV-WA was established using the optimal primer CSV357F/R and the CSV-CP-Probe(47 bp).The working concentrations of the primer and probe were 0.2 and 0.06μmol·L^(-1),respectively.The reaction conditions were set to 42℃for 5 min.This method could specifically detect SPCSV-WA and had no cross-reaction with other common viruses on sweet potato.The RT-RPA-LFD could detect viruses up to 10^(-4) dilution,whereas RT-PCR could only detect up to 10^(-3) dilution,making the sensitivity of RT-RPA-LFD 10 times higher than that of RT-PCR.Of the 22 field-gathered sweet potato samples tested by RT-PCR,RT-RPA,and RT-RPA-LFD,11 positive samples were consistently found across the three methods.The testing results of 28 test tube seedling samples showed that RT-PCR and

关 键 词：甘薯褪绿矮化病毒西非株系 逆转录酶重组酶聚合酶扩增 侧向流层析 快速检测 甘薯 

分 类 号：S435.31[农业科学—农业昆虫与害虫防治]