基于RT-RPA-LFD等温扩增的西瓜银斑驳病毒快速检测技术的建立及应用

doi:10.16420/j.issn.0513-353x.2025-0196

园艺学报 ›› 2025, Vol. 52 ›› Issue (11): 3079-3089.doi: 10.16420/j.issn.0513-353x.2025-0196

基于RT-RPA-LFD等温扩增的西瓜银斑驳病毒快速检测技术的建立及应用

李文扬¹, 修筠清¹, 徐志红¹^,², 王敏³, 古勤生¹, 康保珊¹, 刘莉铭¹, 赵胜杰¹^,², 玉山江·麦麦提⁴^,^*(), 吴会杰¹^,^*()

¹ 中国农业科学院郑州果树研究所，河南省果树瓜类生物学重点实验室，郑州 450009
² 三亚中国农业科学院国家南繁研究院，海南三亚 572024
³ 海南省农业科学院蔬菜研究所，海口 571100
⁴ 新疆维吾尔自治区农业科学院植物保护研究所，农业农村部西北荒漠绿洲作物有害生物综合治理重点实验室，国家植物保护库尔勒观测实验站，新疆农业生物安全重点实验室，乌鲁木齐 830091

收稿日期:2025-06-20 修回日期:2025-10-26 出版日期:2025-11-26 发布日期:2025-11-26
通讯作者:
^* E-mail：yusanjan418@163.com，
wuhuijie@caas.cn
基金资助:
新疆维吾尔自治区重大专项(2023A02009)

Establishment and Application of a Rapid Detection Technique for Watermelon Silver Mottle Virus Based on RT-RPA-LFD

LI Wenyang¹, XIU Junqing¹, XU Zhihong¹^,², WANG Min³, GU Qinsheng¹, KANG Baoshan¹, LIU Liming¹, ZHAO Shengjie¹^,², Yushanjiang · Maimaiti⁴^,^*(), WU Huijie¹^,^*()

¹ Zhengzhou Fruit Research Institute， Chinese Academy of Agricultural Sciences，Henan Key Laboratory Fruit and Cucurbit Biology， Zhengzhou 450009， China
² National Nanfan Research Institute（Sanya）， Chinese Academy of Agricultural Sciences，Sanya， Hainan 572024， China
³ The Vegetable Research Institute， Hainan Academy of Agricultural Sciences， Haikou 571100， China
⁴ Key Laboratory of Integrated Pest Management on Crops in Northwestern Oasis， Ministry of Agriculture and Rural Affairs，National Plant Protection Scientific Observation and Experiment Station of Korla，Xinjiang Key Laboratory of Agricultural Biosafety，Institute of Plant Protection，Xinjiang Uygur Autonomous Region Academy of Agricultural Sciences， Urumqi 830091， China

Received:2025-06-20 Revised:2025-10-26 Published:2025-11-26 Online:2025-11-26

摘要/Abstract

摘要：

建立了一种基于逆转录酶重组酶聚合酶扩增（reverse transcriptase recombinase polymerase amplification，RT-RPA）结合侧向流层析（lateral flow dipstick，LFD）试纸条技术的西瓜银斑驳病毒（watermelon silver mottle virus，WSMoV）快速检测方法。针对WSMoV的L基因保守序列设计特异性引物和探针，通过优化反应体系和条件，确定RT-RPA-LFD最佳引物浓度为0.1 μmol · L^-1、探针浓度为0.06 μmol · L^-1、反应温度为37 ℃和时间12 min。该方法对WSMoV具有高度特异性，与黄瓜绿斑驳花叶病毒（cucumber green mottle mosaic virus，CGMMV）、小西葫芦黄花叶病毒（zucchini yellow mosaic virus，ZYMV）、新德里番茄曲叶病毒（tomato leaf curl new delhi virus，ToLCNDV）、西瓜花叶病毒（watermelon mosaic virus，WMV）、黄瓜花叶病毒（cucumber mosaic virus，CMV）和甜瓜黄斑病毒（melon yellow spot virus，MYSV）等常见西甜瓜病毒无交叉反应；灵敏度与RT-PCR一致，最低检测限达总RNA的10^-4稀释度。对田间32份西甜瓜样品进行检测验证，RT-RPA-LFD与RT-PCR均检出14份阳性，结果一致。本研究建立的RT-RPA-LFD方法反应温度恒定，扩增时间约12 min，结合侧向层析检测，整个过程在20 min内完成，较传统RT-PCR（约2 ~ 3 h）更为简便快捷，且检测结果可视，可用于WSMoV的田间现场快速检测。

关键词: 西瓜, 西瓜银斑驳病毒, RT-RPA-LFD, 可视化检测

Abstract:

A rapid detection method for watermelon silver mottle virus（WSMoV）was developed in this study based on reverse transcriptase recombinase polymerase amplification（RT-RPA）combined with a lateral flow dipstick（LFD）assay. Specific primers and a probe were designed targeting the conserved region of the WSMoV L gene. The reaction system and conditions were optimized，and the optimal parameters were determined as follows：primer concentration 0.1 μmol · L^-1，probe concentration 0.06 μmol · L^-1，reaction temperature 37 ℃，and reaction time 12 min. The established RT-RPA-LFD assay showed high specificity for WSMoV，with no cross-reactivity to other common cucurbit viruses，including cucumber green mottle mosaic virus（CGMMV），zucchini yellow mosaic virus（ZYMV），tomato leaf curl new delhi virus（ToLCNDV），watermelon mosaic virus（WMV），cucumber mosaic virus（CMV），and melon yellow spot virus（MYSV）. The sensitivity of the assay was comparable to that of conventional RT-PCR，with a detection limit of 10^-4 dilution of total RNA. Among 32 field-collected watermelon and melon samples，both RT-RPA-LFD and RT-PCR detected 14 positive samples，showing complete consistency between the two methods. The RT-RPA-LFD assay operates under a constant temperature of 37 ℃ and provides visual results within approximately 20 min，which is much faster and simpler than traditional RT-PCR（2-3 h）. This method enables rapid，sensitive，and visual detection of WSMoV and offers a reliable tool for on-site diagnosis and field surveillance of the virus.

Key words: watermelon, watermelon silver mottle virus, RT-RPA-LFD, visualization detection

李文扬, 修筠清, 徐志红, 王敏, 古勤生, 康保珊, 刘莉铭, 赵胜杰, 玉山江·麦麦提, 吴会杰. 基于RT-RPA-LFD等温扩增的西瓜银斑驳病毒快速检测技术的建立及应用[J]. 园艺学报, 2025, 52(11): 3079-3089.

LI Wenyang, XIU Junqing, XU Zhihong, WANG Min, GU Qinsheng, KANG Baoshan, LIU Liming, ZHAO Shengjie, Yushanjiang · Maimaiti, WU Huijie. Establishment and Application of a Rapid Detection Technique for Watermelon Silver Mottle Virus Based on RT-RPA-LFD[J]. Acta Horticulturae Sinica, 2025, 52(11): 3079-3089.

图/表 10

表1 供筛选的RT-PCR及RT-RPA引物序列

Table 1 RT-PCR and RT-RPA Primer Sequences for Screening

体系 System	引物/探针 Primer/Probe	引物和探针序列（5′-3′） Primer and probe sequence	产物长度/bp Length
RT-PCR	WS174F	GATATATGATTGGAAAGTCTCTGT	174
	WS174R	GACATGTTTCCACTTGGGTT
	WS337F	TTTACAAACAACCTCCAGGAA	337
	WS337R	TCCATTTTTGAGAGTTTTCAGT
	WS295F	TGGTCTGCATCAGATTTAAC	295
	WS295R	CACAAGAATGGTAGACAGAT
	WS536F	CTGATAGAGACAGAAATGGTG	536
	WS536R	CCTATGATCACAGAAGAGAATAATAT
	WS261F	GAAAATGATATGTCAGGAGGAA	261
	WS261R	ACCAGAGTCATTGAACATTTC
RT-RPA	WS1F	AGAGCAATCGTGCAACAATAAGAATATCAGT	247
	WS1R	CTTTGATGGTATTTAGACTTCATGTCCTCAA
	WS2F	GATATATGATTGGAAAGTCTCTGTTGATACCAT	174
	WS2R	GACATGTTTCCACTTGGGTTCAATATAACAA
	WS3F	TGGTCTGCATCAGATTTAACCTATAAATACA	301
	WS3R	5′-biotin-TCATTGCACAAGAATGGTAGACAGATGATAA
	WS3P	5′-FAM-AGAATACGGGTCTTATGGGACTGCTTTATCT[THF]TATTAACAGACAATTTA-C3-spacer

表2 RT-RPA-LFD反应体系组合

Table 2 Combinations of the reaction system of RT-RPA-LFD

编号 Number	引物与探针浓度/（μmol · L^-1）Primer and probe concentration			镁离子浓度/（μmol · L^-1） Mg²⁺ concentration
编号 Number	WS3F（正向引物Forward primer）	WS3R（反向引物Reverse primer）	WS3P（探针Probe）	镁离子浓度/（μmol · L^-1） Mg²⁺ concentration
A-1	0.100	0.100	0.0600	21 000
A-2	0.025	0.025	0.0125	21 000
A-3	0.010	0.010	0.0060	21 000
B-1	0.100	0.100	0.0600	10 500
B-2	0.025	0.025	0.0125	10 500
B-3	0.010	0.010	0.0060	10 500

表3 用于特异性检测的引物列表

Table 3 Table of Primers for Specificity Detection

目标病毒Target virus	引物/探针Primer/Probe	序列（5′-3′）Primer and Probe Sequence	产物长度/bp Length
WsMoV	WS1352F	CGTGCAACAATAAGAATATC	1 352
WsMoV	WS1352R	ATATTAAAAGCACTTTCAATACT
CGMMV	CGF	CAACAAATCAACAACCAACG	985
CGMMV	CGR	AAGTACTCAGACTTCGATTT
ToLCNDV	ToLF	TTCATCCAGATATTCCTTA	949
ToLCNDV	ToLR	GGCTTCGCCTCGTCGTTTTA
MYSV	MYSV F	AGAGCAATCACGGTATCAA	2 120
MYSV	MYSV R	AAAGAGAGAAGGAGAGAGAGA
ZYMV	ZYMV F	TTCTCCAATCACTTCGTTTAC	1 928
ZYMV	ZYMV R	TTGGATCACCAGAAGCTCCTAT
WMV	WMV F	TGAGGAGGAATATGAGGTGG	1 407
WMV	WMV R	AACCGAAGAGCCTTTCATC
CMV	CMV F	ATAGACCGTGGGTCTTATTA	353
CMV	CMV R	TCCGTCTCGAGCGCATCGTC

图1 WSMoV RT-RPA-LFD的检测特异性 A：RT-PCR；B：RT-RPA-LFD；CK：阴性对照。下同

Fig. 1 RT-RPA-LFD specificity for WSMoV detection A：RT-PCR；B：RT-RPA-LFD；CK：Negative control. The same below

图2 WSMoV的RT-RPA检测引物筛选 CK为阴性对照，来源于经RT-PCR检测确认未感染WSMoV的田间西瓜叶片样本

Fig. 2 Screening of RT-RPA primers for WSMoV CK served as the negative control and was derived from field-collected watermelon leaf samples confirmed to be free of WSMoV infection by RT-PCR

图3 不同引物与探针浓度组合在两种镁离子浓度条件下的筛选结果 A-1、A-2等详情见表2

Fig. 3 Screening results of different combinations of primer and probe concentrations under two magnesium ion concentration conditions A-1，A-2 etc. are described in detail in Table 2

图4 RT-RPA-LFD检测体系反应温度的优化

Fig. 4 Optimization of reaction temperature of RT-RPA-LFD detection method

图5 RT-RPA-LFD检测体系反应时间的优化

Fig. 5 Optimization of reaction time of RT-RPA-LFD detection method

图6 WSMoV的RT-PCR（A）和RT-RPA-LFD（B）的灵敏度检测

Fig. 6 Sensitivity of RT-PCR（A）and RT-RPA-LFD（B）for WSMoV detection

图7 田间西甜瓜样品WSMoV检测结果 A：RT-PCR；B：RT-RPA-LFD；1 ~ 32：检测样品；P：阳性对照

Fig. 7 WSMoV detection results of field watermelon and melon samples A：RT-PCR；B：RT-RPA-LFD. 1-32：Tested sample；P：Positive control

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基于RT-RPA-LFD等温扩增的西瓜银斑驳病毒快速检测技术的建立及应用

Establishment and Application of a Rapid Detection Technique for Watermelon Silver Mottle Virus Based on RT-RPA-LFD

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基于RT-RPA-LFD等温扩增的西瓜银斑驳病毒快速检测技术的建立及应用

Establishment and Application of a Rapid Detection Technique for Watermelon Silver Mottle Virus Based on RT-RPA-LFD

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