西番莲夜来香花叶病毒RT-RPA-LFD快速检测方法的建立  

Establishment of RT-RPA-LFD Method for Rapid Detection of Telosma Mosaic Virus in Passiflora

作　　者：丁慧娇 王皓 鲍方维 孙庚 庞婧珺 范在丰 赵文军[3] 周涛 DING Huijiao;WANG Hao;BAO Fangwei;SUN Geng;PANG Jingjun;FAN Zaifeng;ZHAO Wenjun;ZHOU Tao(College of Plant Protection,China Agricultural University,Beijing 100193,China;Sanya Institute,China Agricultural University,Sanya,Hainan 572025,China;Center for Biosafety,Chinese Academy of Inspection and Quarantine,Sanya,Hainan 572019,China)

机构地区：[1]中国农业大学植物保护学院,北京100193 [2]中国农业大学三亚研究院,海南三亚572025 [3]三亚中国检科院生物安全中心,海南三亚572019

出　　处：《园艺学报》2024年第11期2701-2709,共9页Acta Horticulturae Sinica

基　　金：海南省重点研发项目(ZDYF2024XDNY276);中国农业大学三亚研究院引导资金项目(SYND-2022-32)。

摘　　要：根据西番莲上发生的夜来香花叶病毒(telosma mosaic virus,TeMV)的外壳蛋白(coat protein,CP)基因设计引物和探针,将重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)和侧流层析试纸条(lateral flow dipstick,LFD)相结合,建立了一种快速检测TeMV的方法,对该检测方法中的反应时间、温度、探针浓度、引物浓度进行了优化,并检测了该方法的特异性和灵敏度。试验结果表明,RT-RPA-LFD检测体系的最佳反应条件是温度37℃、反应时间13 min、探针浓度5μmol·L^(-1)、引物浓度10μmol·L^(-1)。该方法能够特异性检测TeMV,对含有TeMV cp质粒DNA的最低检测限度为5.125×10^(-4)ng·μL^(-1),比普通PCR检测灵敏10000倍。该方法灵敏度高、特异性强,能够用于田间西番莲样品的检测。Primers and probe were designed according to the coat protein(CP)gene of telosma mosaic virus(TeMV)on passion fruit,and recombinase polymerase amplification(RPA)was combined with lateral flow dipstick(LFD)to establish a method for rapid detection of TeMV.The reaction time,temperature,probe concentration and primers concentration of the method were optimized,and then the specificity and sensitivity of the metho also were examined.The experimental results show that the optimal reaction temperature was 37℃,optimal reaction time was 13 min,optimal probe concentration was 5μmol·L^(-1),and the optimal primers concentration was 10μmol·L^(-1).The method can specifically detect TeMV,and the minimum detection limit could reach to 5.125×10^(-4)ng·L^(-1)for plasmids containing TeMV cp,which is ten thousand times more sensitive than ordinary PCR detection.This method has high sensitivity and specificity,which can be used to detect passion fruit samples in the field.

关 键 词：西番莲 夜来香花叶病毒 重组酶聚合酶扩增 侧流层析试纸条 检测 可视化 

分 类 号：S667.9[农业科学—果树学]