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

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

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (11): 3079-3089.doi: 10.16420/j.issn.0513-353x.2025-0196

• New Technology and New Method • Previous Articles Next Articles

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^,^*(), and 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 Online:2025-11-26 Published:2025-11-26
Contact: Yushanjiang · Maimaiti, and WU Huijie

Abstract

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

LI Wenyang, XIU Junqing, XU Zhihong, WANG Min, GU Qinsheng, KANG Baoshan, LIU Liming, ZHAO Shengjie, Yushanjiang · Maimaiti, and 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.

Figures/Tables 10

References 34

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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

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

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

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

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

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

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