甜瓜种子中黄瓜花叶病毒和小西葫芦黄花叶病毒的RT-LAMP可视化检测方法

doi:10.11924/j.issn.1000-6850.casb20190800499

中国农学通报 ›› 2020, Vol. 36 ›› Issue (20): 107-113.doi: 10.11924/j.issn.1000-6850.casb20190800499

所属专题：园艺

甜瓜种子中黄瓜花叶病毒和小西葫芦黄花叶病毒的RT-LAMP可视化检测方法

袁英哲¹, 罗明¹^,², 韩剑¹^,²(), 潘亚南¹

¹新疆农业大学农学院,乌鲁木齐 830052
²新疆自治区高校农林有害生物监测与安全防控重点实验室,乌鲁木齐 830052

收稿日期:2019-08-04 修回日期:2020-04-23 出版日期:2020-07-15 发布日期:2020-07-20
通讯作者: 韩剑
作者简介:袁英哲,男,1994年出生,河南人,在读硕士,研究方向为植物病害病原学。通信地址：830052 新疆乌鲁木齐市沙依巴克区农大东路311号新疆农业大学农学院,E-mail：1540719097@qq.com。
基金资助:
农业部西北荒漠绿洲作物有害生物综合治理重点实验室开放基金课题“‘一年双收’新种植模式下新疆甜瓜病毒病毒原鉴定、分子变异及主要传播源研究”(KFJJ20150103);国家级大学生创新项目“新疆甜瓜病毒病主要病原病毒环介导等温扩增快速检测技术研究”(201610758059)

Development of a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Visual Detection of Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus in Melon Seeds

Yuan Yingzhe¹, Luo Ming¹^,², Han Jian¹^,²(), Pan Yanan¹

¹College of Agriculture, Xinjiang Agricultural University, Urumqi 830052
²Key Laboratory of Prevention and Control for Pest Monitoring and Security of Autonomous Universities in Xinjiang, Urumqi 830052

Received:2019-08-04 Revised:2020-04-23 Online:2020-07-15 Published:2020-07-20
Contact: Han Jian

摘要/Abstract

摘要：

本研究旨在针对甜瓜种子中携带的黄瓜花叶病毒(CMV)和小西葫芦黄花叶病毒(ZYMV),建立可视化环介导等温扩增检测方法。根据CMV和ZYMV外壳蛋白(CP)基因的保守序列,分别设计并筛选出2套特异性环介导等温扩增(LAMP)引物,通过对反应体系中主要影响因素的优化,建立适用于快速检测CMV和ZYMV的RT-LAMP检测方法。对优化后的2种LAMP检测方法进行特异性和灵敏度验证。结果表明,建立的2种LAMP检测方法分别能够特异性地检测出CMV和ZYMV,检测灵敏度较常规RT-PCR分别高出10倍和100倍,并且可通过肉眼观察直接对反应结果进行判定。该方法的建立为基层及现场快速检测甜瓜种子的带毒情况提供了新方法。

关键词: 甜瓜种子, 黄瓜花叶病毒, 小西葫芦黄花叶病毒, 反转录环介导等温扩增, 可视化检测

Abstract:

The paper aims to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and visual detection of cucumber mosaic virus (CMV) and zucchini yellow mosaic virus (ZYMV) in melon seeds. Two sets of LAMP primers were designed according to the coat protein (CP) gene of CMV and ZYMV. Based on the optimization of reaction conditions, RT-LAMP detection method was developed and verified for specificity and sensitivity. It is suggested that the 2 kinds of LAMP method have specific amplification action respectively for CMV or ZYMV. The sensitivity for the detection of CMV and ZYMV is 10 and 100 times higher than that of the conventional PCR method. The detection result could be judged by visual color change without requiring complex equipment. The method is suitable for detection in field condition and could provide a novel rapid detection approach for CMV and ZYMV in melon seeds.

Key words: melon seeds, cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV), reverse transcription loop-mediated isothermal amplification (RT-LAMP), visual detection

中图分类号:

S436.629

袁英哲, 罗明, 韩剑, 潘亚南. 甜瓜种子中黄瓜花叶病毒和小西葫芦黄花叶病毒的RT-LAMP可视化检测方法[J]. 中国农学通报, 2020, 36(20): 107-113.

Yuan Yingzhe, Luo Ming, Han Jian, Pan Yanan. Development of a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Visual Detection of Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus in Melon Seeds[J]. Chinese Agricultural Science Bulletin, 2020, 36(20): 107-113.

图/表 6

名称	序列(5′→3′)
CMV-F3	TCTACCGTGTGGGTGACA
CMV-B3	GAGCACGGCGTACTTTCG
CMV-FIP	CCGTCCGCGAACATAGCAGAGTTTTGTCCGTAAAGTTCCTGCCTC
CMV-BIP	GCTGCATCTGGAGTCCAAGCCTTTTTGTCGCCAATATCAGCGC
ZYMV-F3	CTCCGGCTCAGGTGAGAA
ZYMV-B3	ATGAGACGCTCGTGTGTTG
ZYMV-FIP	GACGCGGCACAATTTTCCCATTTTTGCAGCTGTCACGAAGGAC
ZYMV-BIP	ATGTCACTGCCACGCGTGAATTTTCGGCTTATACTCCAGCAAGT

表1. LAMP引物序列

图1. RT-LAMP和RT-PCR检测CMV灵敏度对比试验 A为LAMP产物琼脂糖凝胶电泳检测,B为RT-PCR检测,C为LAMP可视化检测,M为100 bp Ladder,1~8分别为1×101~1×108倍cDNA稀释浓度

图2. RT-LAMP和RT-PCR检测ZYMV灵敏度对比试验 A为LAMP产物琼脂糖凝胶电泳检测,B为RT-PCR检测,C为LAMP可视化检测,M为100 bp Ladder,1~8分别为1×101~1×108倍cDNA稀释浓度

图3. CMV-LAMP特异性的琼脂糖凝胶电泳与可视化检测结果 A为琼脂糖凝胶电泳检测,B为可视化检测,M为100bp Ladder,1为健康种子,2~3为CMV,4为WMV,5为SqMV,6为MNSV,7为ZYMV

图4. ZYMV-LAMP特异性的琼脂糖凝胶电泳与可视化检测结果 A为琼脂糖凝胶电泳检测,B为可视化检测,M为100 bp Ladder,1为健康种子,2~3为ZYMV,4为WMV-2,5为SqMV,6为MNSV,7为CMV

品种	检测数/粒	CMV检出率/%		ZYMV检出率/%
品种	检测数/粒	RT-LAMP	RT-PCR	RT-LAMP	RT-PCR
优密17号	10	20	20	80	70
春潇2号	10	20	10	70	70
欣丰密	10	0	0	40	30
楼兰17号	10	0	0	70	50

表2. 甜瓜种子带毒的检测结果

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甜瓜种子中黄瓜花叶病毒和小西葫芦黄花叶病毒的RT-LAMP可视化检测方法

Development of a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Visual Detection of Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus in Melon Seeds

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