蚕豆萎蔫病毒2的RT-LAMP检测方法的建立  

Establishment of RT-LAMP rapid detection method for broad bean wilt virus 2

作　　者：秦艳红[1] 王凤丽 张重义[3] 刘红彦[1] 刘玉霞[1] 高素霞[1] 文艺[1] 李绍建[1] 刘永康 张德胜[1] 鲁书豪 赵正伟 王飞[1] 鲁传涛[1] QIN Yanhong;WANG Fengli;ZHANG Zhongyi;LIU Hongyan;LIU Yuxia;GAO Suxia;WEN Yi;LI Shaojian;LIU Yongkang;ZHANG Desheng;LU Shuhao;ZHAO Zhengwei;WANG fei;LU Chuantao(Institute of Plant Protection,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Wenxian Agriculture Science Research Institute,Wenxian,475004,China;Fujian Agriculture and Forestry University,Fuzhou,350002,China)

机构地区：[1]河南省农业科学院植物保护研究所,郑州450002 [2]温县农业科学研究所,温县475004 [3]福建农林大学,福州350002

出　　处：《植物病理学报》2024年第6期1236-1243,共8页Acta Phytopathologica Sinica

基　　金：国家中药材产业技术体系(CARS-21);河南省中药材产业技术体系(HARS-22-11-Z1);河南省农业科学院基本科研业务费项目(2023ZC051)。

摘　　要：蚕豆萎蔫病毒2(broad bean wilt virus 2,BBWV 2)属于蚕豆病毒属(Fabavirus),是地黄和山药上的重要病毒之一,严重影响这些中药材的产量和品质。本研究建立了BBWV 2的环介导恒温扩增(reverse transcription loop-mediated isothermal amplification, RT-LAMP)检测方法。根据BBWV 2基因组RNA2上的小外壳蛋白(small coat protein, SCP)基因序列的保守区设计引物,对RT-LAMP的反应温度和时间进行了优化,最佳反应条件为60~65℃反应60 min对其灵敏度、特异性和准确性进行了检测,RT-LAMP方法的灵敏度是常规PCR的10倍,为6.47×10^(3)拷贝·μL^(-1),可特异地检测出BBWV 2,对田间地黄、白术和白芷检测的准确性高于常规PCR,对山药检测的准确性度与常规PCR一致。本研究建立的RT-LAMP是一种特异、灵敏、快速、方便的BBWV 2检测方法,适用于基层科研单位对该病毒的准确检测和诊断。Broad bean wilt virus 2(BBWV 2),a member of the genus Fabavirus,is one of the important viruses infecting Rehmannia glutinosa and Dioscorea oppositifolia.This virus seriously affects the yield and quality of these medicinal crops.In this study,the detection method of reverse transcription loop-mediated isothermal amplification(RT-LAMP)for BBWV 2 was established.The primers were designed according to the conserved region of small coat protein(SCP)gene sequence of BBWV 2 RNA2 segments.The amplification temperature and time of the RT-LAMP were optimized and the optimal reaction condition was 60-65℃ within 60 min.The sensitivity,specificity and accuracy were demonstrated.The detection limit of RT-LAMP was 10 times higher than that of the conventional PCR,and it can detect virus at 6.47×10^(3) copies·μL^(-1).This method can specifically detect BBWV 2 and the accuracy of RT-LAMP was higher than conventional PCR used for field samples of Rehmannia Atractylodes macrocephala and Angelica dahurica.The delection rate was consistent with that of conventional PCR for Dioscorea samples.The RT-LAMP established in this study is a specific,sensitive,rapid and convenient method for BBWV 2 detection and is suitable for use in grass-roots scientific research for the accurate detection and diagnosis of BBWV 2.

关 键 词：山药和地黄 蚕豆萎蔫病毒2 逆转录环介导恒温扩增 快速检测 

分 类 号：S432.44[农业科学—植物病理学]