甜瓜内源病毒CmEV的TaqMan RT-qPCR检测方法研究

doi:10.16420/j.issn.0513-353x.2021-0620

园艺学报 ›› 2022, Vol. 49 ›› Issue (11): 2471-2478.doi: 10.16420/j.issn.0513-353x.2021-0620

甜瓜内源病毒CmEV的TaqMan RT-qPCR检测方法研究

赵立群¹, 张晓飞², 邱艳红²^,³^,^*(), 刘慧², 张建²^,³, 杨静静², 张海军²^,³, 徐秀兰²^,³, 温常龙²^,³^,^*()

¹北京市农业技术推广站，北京 100029
²农业农村部华北地区园艺作物生物学与种质创制重点实验室，北京市农林科学院蔬菜研究所，北京 100097
³农业农村部蔬菜种子质量监督检验测试中心，北京 100097

收稿日期:2022-01-19 修回日期:2022-03-24 出版日期:2022-11-25 发布日期:2022-11-25
通讯作者: 邱艳红,温常龙 E-mail:qiuyanhong@nercv.org;wenchanglong@nercv.org
基金资助:
国家重点研发计划项目(2017YFD0102004)

Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method

ZHAO Liqun¹, ZHANG Xiaofei², QIU Yanhong²^,³^,^*(), LIU Hui², ZHANG Jian²^,³, YANG Jingjing², ZHANG Haijun²^,³, XU Xiulan²^,³, WEN Changlong²^,³^,^*()

¹Beijing Agricultural Extension Station，Beijing 100029，China
²Key Laboratory of Biology and Genetics Improvement of Horticultural Crops（North China），Institute of Vegetable Science，Beijing Academy of Agricultural and Forestry Sciences，Beijing 100097，China
³Supervision，Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs，Beijing 100097，China

Received:2022-01-19 Revised:2022-03-24 Online:2022-11-25 Published:2022-11-25
Contact: QIU Yanhong,WEN Changlong E-mail:qiuyanhong@nercv.org;wenchanglong@nercv.org

摘要/Abstract

摘要：

甜瓜内源病毒（cucumis melo endornavirus，CmEV）是近年来发现的寄主广泛、发病率高、种传率高的重要病毒。利用CmEV基因组的序列信息设计特异性引物和探针，建立基于探针法的实时荧光定量PCR（Real-Time Fluorescent Quantitative PCR with TaqMan probes，TaqMan RT-qPCR）的检测方法。该方法可以检测出1 × 10^-2ng · μL^-1的RNA和1.8 × 10³copies · μL^-1的质粒，灵敏度是普通PCR的100倍。利用该方法检测了在北京、山东、辽宁、海南瓜类种苗主产区的甜瓜、西瓜、黄瓜与南瓜砧木11份种苗带毒情况，发现山东和海南各有1份甜瓜种苗携带CmEV。本研究建立的CmEV TaqMan RT-qPCR方法特异性好、灵敏度高，可为开展病毒精准鉴定、科学防控以及从源头遏制病毒传播提供技术支撑。

关键词: 甜瓜内源病毒, TaqMan探针法荧光实时定量PCR, 葫芦科作物, 种苗带毒

Abstract:

Cucumis melo endornavirus is an important plant virus that infects a wide range of hosts，occurs popularly and transmits highly in seeds. One set of specific primers and probes were designed according to the conserved sequence of CmEV genome，and a detecting method of CmEV was established using the Real-Time Fluorescent Quantitative PCR with TaqMan probes（TaqMan RT-qPCR）. This detection method could detect the virus in 1 × 10^-2ng · μL^-1 RNA samples and 1.8 × 10³ copies · μL^-1 plasmids，the sensitivity of which was about one hundred times higher than traditional RT-PCR. We used the CmEV TaqMan RT-qPCR technique to detect 11 seedling lots of Cucumis melo，Citrullus lanatus，Cucumis sativus，Cucurbita moschata stock that randomly sampled from the main cucurbit industries of Beijing，Shandong，Liaoning and Hainan provinces. It was found that Cucumis melo seedling from Shandong and Hainan industries carried CmEV. The novel TaqMan RT-qPCR method was proved to be efficient in testing CmEV，which could be a technical support for precisely detecting and scientific controlling of this virus，and could help preventing the virus spread from the source.

Key words: cucumis melo endornavirus, TaqMan qPCR, cucurbits crops, seedling-borne disease

中图分类号:

S652

赵立群, 张晓飞, 邱艳红, 刘慧, 张建, 杨静静, 张海军, 徐秀兰, 温常龙. 甜瓜内源病毒CmEV的TaqMan RT-qPCR检测方法研究[J]. 园艺学报, 2022, 49(11): 2471-2478.

ZHAO Liqun, ZHANG Xiaofei, QIU Yanhong, LIU Hui, ZHANG Jian, YANG Jingjing, ZHANG Haijun, XU Xiulan, WEN Changlong. Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method[J]. Acta Horticulturae Sinica, 2022, 49(11): 2471-2478.

图/表 7

表1 检测种苗携带CmEV的主要瓜类种苗信息

Table 1 The information of Cucurbits crops seedlings for detection of CmEV

序号 Number	种苗 Seedling	产地 Area	序号 Number	瓜类种苗 Seedling	产地 Area
1	甜瓜 Melon	北京 Beijing	7	黄瓜Cucumber	北京 Beijing
2	甜瓜 Melon	山东 Shandong	8	黄瓜Cucumber	山东 Shandong
3	甜瓜 Melon	辽宁 Liaoning	9	黄瓜Cucumber	辽宁 Liaoning
4	甜瓜 Melon	海南 Hainan	10	南瓜砧木Pumpkin stock	北京 Beijing
5	西瓜 Watermelon	北京 Beijing	11	南瓜砧木Pumpkin stock	辽宁 Liaoning
6	西瓜 Watermelon	山东Shandong

表2 CmEV引物序列信息

Table 2 The sequence of primers used for detection of the CmEV

引物 Primer	序列（5′-3′） Sequence	扩增片段/bp Amplified fragement
CmEV-qRT	P：CATACGCTTCGAAGACCCTCTTGCT	160
	F：GAGATACTTGTGGGTCAGTGGCAG
	R：GTCGGAAATTGGTTTATGTGGTG
CmEV-500	F：GGTAATGGCTTCATTCCCCATTAC	500
CmEV-RT	F：CGCGGAAATTGCACTTCCGAGAT	755
CmEV-RT	R：CTAGCAACTTGTTTGCTGGCACATG	755

图1 CmEV TaqMan RT-RqPCR检测特异性分析

Fig. 1 Specificity of TaqMan-based RT-qPCR for detection CmEV

图2 常规RT-PCR检测结果

Fig. 2 The amplified results of RT-PCR detection

图3 CmEV的TaqMan RT-qPCR和常规RT-PCR的灵敏度检测 A：7个稀释度TaqMan RT-qPCR扩增曲线;B：TaqMan RT-qPCR的标准曲线;C：常规RT-PCR灵敏度测试。

Fig. 3 The detection sensitivity of TaqMan RT-qPCR and conventional RT-PCR of CmEV A：Amplification curves of TaqMan RT-qPCR of seven serial dilutions，B：TaqMan RT-qPCR standard quantification curve，C：Sensitivity of the conventional PCR assay.

图4 CmEV的TaqMan qPCR和常规PCR的灵敏度检测 A：7个稀释度TaqMan qPCR扩增曲线;B：TaqMan qPCR的标准曲线;C：常规PCR灵敏度测试。

Fig. 4 The detection sensitivity of TaqMan qPCR and conventional RT-PCR of CmEV A：Amplification curves of TaqMan qPCR of seven ten-fold serial dilutions，B：TaqMan qPCR standard quantification curve，C：Sensitivity of the conventional PCR assay.

图5 主要瓜类蔬菜种苗携带CmEV分析

Fig. 5 Detection of CmEV in major cucurbits crops seedlings

参考文献 21

[1]	Chai Ali,Patiguli,Guo Weitao,Shi Yanxia,Xie Xuewen,Xi Xianmei,Li Baoju. 2019. Development and application of quantitative PCR for detection of Pseudomonas syringae pv. tomato. Acta Horticulturae Sinica, 46 (1):182-192. (in Chinese)
	柴阿丽, 帕提古丽, 郭威涛, 石延霞, 谢学文, 席先梅, 李宝聚. 2019. 番茄细菌性斑点病菌实时荧光定量PCR检测方法的建立及应用. 园艺学报, 46 (1):182-192. doi: 10.16420/j.issn.0513-353x.2018-0294
[2]	Cheng Yingchao, Kang Hua-jun, Shi Yanxia, Chai Ali, Zhang Hongjie, Xie Xuewen, Li Baoju. 2018. Development and application of real-time fluorescent quantitative PCR for detection of Phytophthora capsici. Acta Horticulturae Sinica 45,( 5):997-1006. (in Chinese)
	程颖超, 康华军, 石延霞, 柴阿丽, 张红杰, 谢学文, 李宝聚. 2018. 辣椒疫霉菌RT-PCR检测技术的建立及应用. 园艺学报, 45 (5) :997-1006.
[3]	Choi G S. 2001. Occurrence of two tobamovirus diseases in cucurbits and control measures in Korea. The Plant Pathology Journal, 17 (5):243-248.
[4]	Coutts R H A. 2005. First report of an endornavirus in the Cucurbitaceae. Virus Genes, 31:361-362.
[5]	Da Costa A C, Leal E, Gill D, Milagres F A D P, Komninakis S V, Brustulin R, Teles M D A R, Lobato M C A B S,das Chagas R T,Abrao M D F N D S,Soares C V D A,Deng X,Delwart E,Sabino E C,Luchs A. 2019. Discovery of Cucumis melo endornavirus by deep sequencing of human stool samples in Brazil. Virus Genes, 55:332-338. doi: 10.1007/s11262-019-01648-0 pmid: 30915664
[6]	Dinant S, Lot H. 1992. Lettuce mosaic virus. Plant Pathology, 41:528-542. doi: 10.1111/j.1365-3059.1992.tb02451.x URL
[7]	Fan Xu-dong, Dong Ya-feng, Zhang Zun-ping, Ren Fang, Hu Guo-jun, Zhu Hong-juan. 2014. Progress on molecular detection of grapevine virus. Acta Horticulturae Sinica, 41 (5):1009-1019. (in Chinese)
	范旭东, 董雅凤, 张尊平, 任芳, 胡国君, 朱红娟. 2014. 葡萄病毒分子检测技术研究进展. 园艺学报, 41 (5):1009-1019.
[8]	Fukuhara T. 2019. Endornaviruses:persistent dsRNA viruses with symbiotic properties in diverse eukaryotes. Virus Genes, 55:165-173. doi: 10.1007/s11262-019-01635-5 URL
[9]	He Zhen, Dong Tingting, Wu Weiwen, Chen Wen, Li Liangjun. 2020. Development and evaluation of a Real-time fluorescent quantitative PCR assay for detection of sweet potato latent virus-lotus in lotus plants. Acta Horticulturae Sinica, 47 (7):1412-1420. (in Chinese)
	贺振, 董婷婷, 吴伟文, 陈雯, 李良俊. 2020. 莲藕中甘薯潜隐病毒实时荧光定量PCR检测技术的建立及应用. 园艺学报, 47 (7):1412-1420.
[10]	Hui Wen-guang, Zhao Ting-chang, Schaad N W, Sun Fu-zai, Wang Jian-rong. 2007. Establishment of rapid detection method for the pathogen of hami melon fruit blotch. Scientia Agricultura Sinica, 40 (11):2495-2501. (in Chinese)
	回文广, 赵廷昌, Schaad N W, 孙福在, 王建荣. 2007. 哈密瓜细菌性果斑病菌快速检测方法的建立. 中国农业科学, 40 (11):2495-2501.
[11]	Maule A J, Wang D. 1996. Seed transmission of plant viruses:a lesson in biological complexity. Trends in Microbiology, 4 (4):153-158. doi: 10.1016/0966-842x(96)10016-0 pmid: 8728609
[12]	Qin Zi-yu, Sun Jian-she, Wang Na, Shao Jian-zhu. 2015. Development of a TaqMan real-time RT-PCR method for detecting Apple stem pitting virus (ASPV). Acta Horticulturae Sinica, 42 (7):1400-1408. (in Chinese)
	秦子禹, 孙建设, 王娜, 邵建柱. 2015. 苹果茎痘病毒TaqMan探针实时荧光定量RT-PCR检测方法的建立. 园艺学报, 42 (7):1400-1408.
[13]	Quito-Avil D F, Peralta E L, Martin R R, Ibarra M A, Alvarez R A, Mendoza A, Insuasti M, Ochoa L. 2014. Detection and occurrence of melon yellow spot virus in Ecuador:an emerging threat to cucurbit production in the region. Eur J Plant Pathol, 140:193-197. doi: 10.1007/s10658-014-0454-1 URL
[14]	Ren Fang, Dong Yafeng, Zhang Zunping, Fan Xudong, Hu Guojun. 2018. Development and application of a quantitative RT-PCR approach fordetection of Grapevine virus A. Acta Horticulturae Sinica, 45 (11):2243-2253. (in Chinese)
	任芳, 董雅凤, 张尊平, 范旭东, 胡国君. 2018. 葡萄病毒 A 实时荧光定量RT-PCR检测技术的建立及应用. 园艺学报, 45 (11):2243-2253.
[15]	Sabanadzovic S, Wintermantel W M, Valverdec R A, McCreight J D, Aboughanem-Sabanadzovic N. 2016. Cucumis melo endornavirus:genome organization,host range and co-divergence with the host. Virus Research, 214:49-58. doi: 10.1016/j.virusres.2016.01.001 pmid: 26805037
[16]	Sun Jie, Wang Wan, Rao Xue-qin,Li Hua-ping. 2016. Development of TaqMan Real-time PCR Assay of Banana bunchy top virus. Acta Horticulturae Sinica, 43 (12):2473-2480. (in Chinese)
	孙洁, 王婉, 饶雪琴, 李华平. 2016. 香蕉束顶病毒实时荧光 PCR 检测方法的建立. 园艺学报, 43 (12):2473-2480.
[17]	Wang Yan-jiao, Cui Tian-tian, Huang Ai-jun, Chen Hong-ming, Li Zhong-an, Zhou Chang-yong, Song Zhen. 2016. Development and application of a quantitative RT-PCR approach for quantification of Citrus vein enation virus. Acta Horticulturae Sinica, 43 (8):1613-1620. (in Chinese)
	王艳娇, 崔甜甜, 黄爱军, 陈洪明, 李中安, 周常勇, 宋震. 2016. 柑橘脉突病毒实时荧光定量RT-PCR检测体系的建立与应用. 园艺学报, 43 (8):1613-1620.
[18]	Wu Hui-Jie, Qin Bi-xia, Chen Hong-yun, Peng Bin, Cai Jian-he, Gu Qin-sheng. 2011. The rate of seed contamination and transmission of cucumber green mottle mosaic virus in watermelon and melon. Scientia Agricultura Sinica, 44 (7):1527-1532. (in Chinese)
	吴会杰, 秦碧霞, 陈红运, 彭斌, 蔡建和, 古勤生. 2011. 黄瓜绿斑驳花叶病毒西瓜、甜瓜种子的带毒率和传毒率. 中国农业科学, 44 (7):1527-1532.
[19]	Zeng R, Xu L H, Gao S G, Song Z W, Gao P, Dai F M. 2020. First report of Cucumis melo endornavirus on Cucumis melo in China. Journal of Plant Pathology, 102:1355. doi: 10.1007/s42161-020-00637-9 URL
[20]	Zhang Mengyan, Zhang Zunping, Ren Fang, Hu Guojun, Fan Xudong, Dong Yafeng. 2020. Establishment and application of a real-time fluorescent quantitative RT-PCR for detection of Grapevine fabavirus. Acta Horticulturae Sinica, 47 (1):187-194.
	张梦妍, 张尊平, 任芳, 胡国君, 范旭东, 董雅凤. 2020. 葡萄蚕豆萎蔫病毒实时荧光定量 RT-PCR 检测方法及应用. 园艺学报, 47 (1):187-194.
[21]	Zhou Jun, Fan Xu-dong,Dong Ya-feng,Zhang Zun-ping,Hu Guo-jun,Ren Fang,Li Zheng-nan. 2016. Development and application of a quantitative RT-PCR approach for quantification of Grapevine fanleaf virus. Acta Horticulturae Sinica, 43 (3):538-548. (in Chinese)
	周俊, 范旭东, 董雅凤, 张尊平, 胡国君, 任芳, 李正男. 2016. 葡萄扇叶病毒实时荧光定量RT-PCR检测方法的建立及应用. 园艺学报, 43 (3):538-548.

甜瓜内源病毒CmEV的TaqMan RT-qPCR检测方法研究

Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 21

相关文章 15

编辑推荐

Metrics

本文评价

访问总数:　当日访问总数:　当前在线人数:

[1]	任凯丽, 孔维萍, 唐桃霞, 程鸿. 基于Lab表色系统的甜瓜果实颜色多样性分析[J]. 园艺学报, 2022, 49(9): 2017-2022.
[2]	窦宏涛, 卢于绒, 武云霞, 杨小振, 刘民强, 周玲, 詹涛. 早熟薄皮甜瓜新品种‘西甜5号’[J]. 园艺学报, 2022, 49(7): 1617-1618.
[3]	任俭, 张娜, 李煜华, 曾红霞, 孙玉宏, 陈伟, 熊建顺, 汤谧. 甜瓜新品种‘M1004’[J]. 园艺学报, 2022, 49(6): 1397-1398.
[4]	李超, 杨英, 陈伟, 郑贺云, 廖新福, 孙玉萍. 西州密系列甜瓜SSR指纹图谱构建及聚类分析[J]. 园艺学报, 2022, 49(3): 622-632.
[5]	盛云燕, 杨丽敏, 戴冬洋, 张佳欣, 王岭, 王迪, 才羿, 田丽美. 甜瓜果柄离区细胞学观察及成熟脱落基因AL3的初步定位[J]. 园艺学报, 2022, 49(2): 341-351.
[6]	施兆荣, 张广荣, 孙述俊, 金梦军, 崔凌霄, 刘彦超, 魏立娟, 梅大海, 杨成德. 甘肃省甜瓜黑斑病菌（Alternaria tenuissima）的分离鉴定[J]. 园艺学报, 2022, 49(2): 427-436.
[7]	曹燕燕, 刁倩楠, 陈幼源, 张永平. 甜瓜新品种‘金蜜一号’[J]. 园艺学报, 2022, 49(2): 467-468.
[8]	程勋辉, 谢钊, 杨中周, 代伟, 戴祖云, 宋江华. 甜瓜新品种‘江淮蜜7号’[J]. 园艺学报, 2021, 48(9): 1841-1842.
[9]	王康, 闫洪朗, 何林池, 魏小云, 陆小强. 薄皮甜瓜新品种‘通甜蜜1号’[J]. 园艺学报, 2021, 48(1): 197-198.
[10]	刘志恒, 侯悦, 胡积祥, 滕晓菲, 黄欣阳, 王世维, 赵廷昌. 辽宁省甜瓜果腐病病原菌鉴定及生物学特性初探[J]. 园艺学报, 2013, 40(1): 89-97.
[11]	张建农;陶永红. 甜瓜新品种‘黄河蜜6号’[J]. 园艺学报, 2006, 33(5): 1168-1168.
[12]	袁昌梅;罗卫红;;张生飞;戴剑锋;金亮. 温室网纹甜瓜发育模拟模型研究[J]. 园艺学报, 2005, 32(02): 262-267.
[13]	李素文;孙德岭;温媛;王公恕;张宝珍;方文惠;赵前程;刘莉莉. 花椰菜新品种‘津品88’[J]. 园艺学报, 2005, 32(02): 371-371.
[14]	马国斌;严秀琴;倪秀红;张　辉. 优质厚皮甜瓜新品种‘明珠1号’[J]. 园艺学报, 2005, 32(02): 373-373.
[15]	徐永阳;徐志红;刘君璞. 厚皮甜瓜新品种‘中甜2号’[J]. 园艺学报, 2005, 32(02): 375-375.

甜瓜内源病毒CmEV的TaqMan RT-qPCR检测方法研究

Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 21

相关文章 15

编辑推荐

Metrics

本文评价

访问总数: 当日访问总数: 当前在线人数:

访问总数:　当日访问总数:　当前在线人数: