西番莲夜来香花叶病毒RT-RPA-LFD快速检测方法的建立

doi:10.16420/j.issn.0513-353x.2023-0701

摘要/Abstract

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5 μmol · L^-1、引物浓度10 μmol · 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10 000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。

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

Abstract:

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 5 μmol · L^-1，and the optimal primers concentration was 10 μmol · L^-1. The method can specifically detect TeMV，and the minimum detection limit could reach to 5.125 × 10^-4 ng · μL^-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.

Key words: Passiflora, telosma mosaic virus, recombinase polymerase amplification, lateral flow dipstick, detection, visualization

丁慧娇, 王皓, 鲍方维, 孙庚, 庞婧珺, 范在丰, 赵文军, 周涛. 西番莲夜来香花叶病毒RT-RPA-LFD快速检测方法的建立[J]. 园艺学报, 2024, 51(11): 2701-2709.

DING Huijiao, WANG Hao, BAO Fangwei, SUN Geng, PANG Jingjun, FAN Zaifeng, ZHAO Wenjun, ZHOU Tao. Establishment of RT-RPA-LFD Method for Rapid Detection of Telosma Mosaic Virus in Passiflora[J]. Acta Horticulturae Sinica, 2024, 51(11): 2701-2709.

图/表 6

图1 RPA引物对在TeMV cp基因序列中的结合位点斜体表示RPA引物。

Fig. 1 Binding sites of RPA primers in the cp gene sequence of TeMV Italics indicate RPA primers.

图2 表现花叶症状的西番莲叶片

Fig. 2 A leaf of Passiflora edulis with mosaic symptoms

图3 RT-RPA-LFD检测体系反应条件的优化 C：质控线；T：检测线。下同。

Fig. 3 Optimization of reaction conditions for RT-RPA-LFD detection system C：Control line；T：Test line. The same below.

图4 RT-PCR检测和RT-RPA-LFD检测TeMV、SCMV、TuMV、PPV、PVMV、CABYV、PLV M：DNA marker。RT-PCR检测TeMV的目标条带分别为398 bp和816 bp。

Fig. 4 RT-PCR detection and RT-RPA-LFD detection of TeMV，SCMV，TuMV，PPV，PVMV，CABYV and PLV M：DNA marker. The target bands of TeMV detected by RT-PCR were 398 bp and 816 bp，respectively.

图5 普通RT-PCR和RT-RPA-LFD检测含有TeMV cp基因的不同浓度的质粒DNA

Fig. 5 Detection of different concentrations TeMV cp-containing plasmids by ordinary RT-PCR and RT-RPA-LFD

图6 采用RT-RPA-LFD和RT-PCR检测西番莲田间样品中的TeMV +：阳性对照；-：阴性对照；1 ~ 14：西番莲样品。

Fig. 6 Passion fruit field samples were detected for TeMV by RT-RPA-LFD and RT-PCR +：Positive control；-：Negative control；1-14：Passion fruit samples.

参考文献 26

[1]	Cerqueira-Silva C B M,Conceição L D H C S,Souza A P,Corrêa R X. 2014. A history of passion fruit woodiness disease with emphasis on the current situation in Brazil and prospects for Brazilian passion fruit cultivation. European Journal of Plant Pathology, 139:261-270.
[2]	Chen Lilang, Yang Tianzhang, Cai Ruping, Lin Xiaoman, Deng Nankang, Che Haiyan, Lin Yating, Kong Xiangyi. 2022. Molecular detection and identification of viruses from Passiflora edulis in Hainan. Acta Horticulturae Sinica, 49 (8):1785-1794. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0418
	陈礼浪, 杨天章, 蔡儒平, 林小漫, 邓南康, 车海彦, 林雅婷, 孔祥义. 2022. 海南西番莲主要病毒种类的分子检测与鉴定. 园艺学报, 49 (8):1785-1794. doi: 10.16420/j.issn.0513-353x.2021-0418
[3]	Chen S S, Yu N N, Yang S H, Zhong B P, Lan H H. 2018. Identification of telosma mosaic virus infection in Passiflora and its impact on phytochemical contents. Virology Journal, 15 (1):1-168.
[4]	Ghosh D K, Kokane S B, Kokane A D, Warghane A J, Motghare M R, Bhose S, Sharma A K, Reddy M K. 2018. Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay(HLB-RPA-LFA)for rapid detection of“Candidatus Liberibacter asiaticus”. PLoS ONE, 13 (12):e0208530.
[5]	Ha C, Coombs S, Revill P A, Harding R M, Vu M, Dale J L. 2008. Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses. Archives of Virology, 153 (1):25-36. doi: 10.1007/s00705-007-1053-7 pmid: 17906831
[6]	Han Tong. 2019. Molecular detection and identification of three woody plant viral diseases in Shenyang[M. D. Dissertation]. Shenyang: Shenyang University. (in Chinese)
	韩彤. 2019. 沈阳三种木本植物病毒病的分子检测与鉴定[硕士论文]. 沈阳: 沈阳大学.
[7]	Huo Danqun, Jiang Lan, Ma Lulu, Hou Changjun, Yang Ping. 2012. Research on passion fruit function and its development progress. Science and Technology of Food Industry, 33:391-395. (in Chinese)
	霍丹群, 蒋兰, 马璐璐, 候长军, 杨平. 2012. 百香果功能研究及其开发进展. 食品工业科技, 33:391-395.
[8]	Kersting S, Rausch V, Bier F F,von Nickisch-Rosenegk M. 2014. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malaria Journal, 13:1-9.
[9]	Li J, Macdonald J,von Stetten F. 2020. Correction: Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. The Analyst, 145 (5):1950-1960.
[10]	Li Wei,Li Chunjian,Zhang Fusuo,Huang Biao. 2022. Research and application progress of passion fruit nutritional quality and functional ingredients. China Agricultural University Journal of Social Sciences Edition, 27 (11):79-92. (in Chinese)
	李巍, 李春俭, 张福锁, 黄彪. 2022. 西番莲营养品质与功能性成分研究及应用进展. 中国农业大学学报, 27 (11):79-92.
[11]	Li Xiaoqin, Zhang Pengyuan, Ren Longhui, Dai Jinran, Wang Jianguang, Chen Suiyun. 2017. Prokaryotic expression of Basella rugose mosaic virus CP gene and preparation of its polyclonal antiserum. Acta Horticulturae Sinica, 44 (10):2001-2007. (in Chinese)
	李小琴, 张鹏远, 任龙辉, 代瑾然, 王建光, 陈穗云. 2017. 落葵皱缩花叶病毒CP基因的原核表达及多克隆抗血清的制备. 园艺学报, 44 (10):2001-2007. doi: 10.16420/j.issn.0513-353x.2016-0826
[12]	Li Xue, Li Jinhai, Wang Lanhua, Qi Duo,Guo Mengmeng,Zhou Xueping,Wu Jianxiang. 2022. Preparation and detection of broad spectrum monoclonal antibodies against three potato viruses belonging to the potyvirus infecting passion fruit. Journal of Agricultural Biotechnology, 30 (9):1845-1854. (in Chinese)
	李雪, 李晋海, 王兰花, 戚朵, 郭萌萌, 周雪平, 吴建祥. 2022. 侵染西番莲的三种马铃薯Y病毒属病毒的广谱单克隆抗体制备及检测应用. 农业生物技术学报, 30 (9):1845-1854.
[13]	Luo Jinshui, Zhou Zhien, Wang Longyan, Chen Zhendong, Wu Zujian, Lin Rongyao. 2019. Occurrence and prevention and control of passion fruit virus disease. Southeast Horticulture, 7 (6):36-40. (in Chinese)
	罗金水, 周知恩, 王隆燊, 陈振东, 吴祖建, 林荣耀. 2019. 西番莲病毒病的发生与防控. 东南园艺, 7 (6):36-40.
[14]	Mckern N M, Strike P M, Barnett O W, Dijkstra J, Shukla D D, Ward C W. 1994. Cowpea aphid borne mosaic virus-Morocco and South African Passiflora virus are strains of the same potyvirus. Archives of Virology, 136:207-217.
[15]	Noveriza R, Suastika G, Hidayat S H, Natsuaki K T. 2016. Molecular cloning and sequencing of telosma mosaic virus(TeMV)causing mosaic disease on patchouli. Plant Pathology Journal, 15 (2):65-74.
[16]	Piepenburg O, Williams C H, Stemple D L, Armes N A. 2006. DNA detection using recombination proteins. Plos Biology, 4 (7):1115-1121.
[17]	Qian Yike, Wei Shuang, Huang Fayu, Chen Yongqing, Chen Weimin. 2021. Establishment of RPA detection method for Diaporthe helianthi. Plant Quarantine, 35 (2):39-43. (in Chinese)
	乾义柯, 魏霜, 黄法余, 陈永青, 陈卫民. 2021. 向日葵茎溃疡病菌RPA检测方法的建立. 植物检疫, 35 (2):39-43.
[18]	Song Y S, Ryu K H. 2011. The complete genome sequence and genome structure of passion fruit mosaic virus. Archives of Virology, 156 (6):1093-1095. doi: 10.1007/s00705-011-0961-8 pmid: 21547441
[19]	Wylie S J, Jones M G. 2011. The complete genome sequence of a passion fruit woodiness virus isolate from Australia determined using deep sequencing,and its relationship to other potyviruses. Archives of Virology, 156 (3):479-482.
[20]	Xie L X, Gao F L, Zheng S, Zhang X Y, Zhang L J, Li T. 2019. Molecular characterization of a new potyvirus infecting passion fruit. Archives of Virology, 164 (7):1903-1906. doi: 10.1007/s00705-019-04251-8 pmid: 30972590
[21]	Xie Lixue, Zhang Lijie, Zhang Xiaoyan, Zheng Shan, Li Tao. 2018. First report of East Asian passiflora virus infecting Passiflora edulis in Fujian,China. Acta Horticulturae Sinica, 45 (8):1587-1594. (in Chinese) doi: 10.16420/j.issn.0513-353x.2018-0116
	谢丽雪, 张立杰, 张小艳, 郑姗, 李韬. 2018. 福建西番莲上首次检出东亚西番莲病毒. 园艺学报, 45 (8):1587-1594. doi: 10.16420/j.issn.0513-353x.2018-0116
[22]	Xie Lixue, Zhang Xiaoyan, Zheng Shan, Zhang Lijie, Li Tao. 2017. Molecular identification and specificity detection of telosma mosaic virus in passion fruit. Scientia Agricultura Sinica, 50:4725-4734. (in Chinese)
	谢丽雪, 张小艳, 郑姗, 张立杰, 李韬. 2017. 侵染西番莲的夜来香花叶病毒的分子鉴定及特异性检测. 中国农业科学, 50:4725-4734. doi: 10.3864/j.issn.0578-1752.2017.24.006
[23]	Yan Jiawen, Yuan Qifeng, Xie Pu, Wang Lijuan, Ma Yuhua, Wang Zhengyuan. 2019. Small RNA sequencing technology was used to detect passionfruit virus in Guizhou. Chinese Journal of Tropical Crops, 40 (8):1577-1584. (in Chinese) doi: 10.3969/j.issn.1000-2561.2019.08.018
	严佳文, 袁启凤, 解璞, 王立娟, 马玉华, 王正媛. 2019. 利用小RNA测序技术检测贵州西番莲病毒. 热带作物学报, 40 (8):1577-1584. doi: 10.3969/j.issn.1000-2561.2019.08.018
[24]	Yang Jun, Nie Fuping, Wang Yu, Shi Meimei, Xie Xiaoqian, Wang Guomin, Li Xianliang, Wu Rui,Zhang Huan,Tang Changjie,Li Yingguo. 2022. Establishment and application of real-time fluorescent recombinase polymerase amplification method for detection of classical swine fever virus. Journal of Anhui Agricultural Sciences, 50 (20):166-169,173. (in Chinese)
	杨俊, 聂福平, 王昱, 史梅梅, 谢晓倩, 王国民, 李贤良, 吴蕊, 张欢, 唐昌杰, 李应国. 2022. 猪瘟病毒实时荧光重组酶聚合酶扩增检测方法的建立及应用. 安徽农业科学, 50 (20):166-169,173.
[25]	Yi Zilin, Xu Weiqing. 2018. Current situation and prospect of passion fruit introduction and cultivation in Hainan. Chinese Journal of Tropical Agriculture, 38:25-28. (in Chinese)
	易籽林, 徐卫清. 2018. 百香果在海南引种栽培现状与展望. 热带农业科学, 38:25-28.
[26]	Zhang Ning, Lin Qinghong, Zeng Xinping, Huang Weinan. 2003. Research progress in passion fruit biotechnology(Review). Subtropical Plant Science, 32:77-80. (in Chinese)
	章宁, 林清洪, 曾新萍, 黄维南. 2003. 西番莲生物技术研究进展(综述). 亚热带植物科学, 32:77-80.