辣椒斑驳病毒RT-LAMP-LFD快速检测方法的建立

doi:10.3969/j.issn.1004-1524.20240711

浙江农业学报 ›› 2026, Vol. 38 ›› Issue (2): 310-316.DOI: 10.3969/j.issn.1004-1524.20240711

辣椒斑驳病毒RT-LAMP-LFD快速检测方法的建立

付康(), 张灿, 赵薇, 马宁, 俞晓平, 孙凯()

中国计量大学生命科学学院, 浙江省生物计量及检验检疫技术重点实验室, 浙江杭州 310018

收稿日期:2024-08-05 出版日期:2026-02-25 发布日期:2026-03-24
作者简介:付康,从事病毒鉴定研究。E-mail:1252544376@qq.com
通讯作者: ^*孙凯,E-mail:sunkai@cjlu.edu.cn

Establishment of a rapid detection method for pepper mottle virus using RT-LAMP-LFD

FU Kang(), ZHANG Can, ZHAO Wei, MA Ning, YU Xiaoping, SUN Kai()

Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China

Received:2024-08-05 Online:2026-02-25 Published:2026-03-24

摘要/Abstract

摘要：

辣椒斑驳病毒(PepMoV),隶属马铃薯Y病毒属(Potyvirus),是侵染辣椒的重要病毒之一。该病毒通过蚜虫以非持久方式传播,对浙江省辣椒产业造成严重威胁。为了实现该病毒的快速检测,本研究建立了一种基于逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification, RT-LAMP)结合横向流动试纸条(lateral flow dipstick, LFD)的检测方法,可实现PepMoV的快速可视化检测。针对PepMoV外壳蛋白(coat protein, CP)基因的保守序列,设计了一套特异性RT-LAMP引物。优化后的RT-LAMP-LFD方法在63 ℃恒温条件下,反应40 min,再进行5 min试纸条显色反应,即可完成样品检测。该方法在特异性和灵敏度方面表现优异,与传统RT-qPCR相比,灵敏度提高了10倍,为PepMoV的快速诊断提供了一种高效、便捷的工具,有望广泛应用于辣椒病害防控。

关键词: 辣椒斑驳病毒, RT-LAMP-LFD, 快速检测, 植物检疫

Abstract:

Pepper mottle virus (PepMoV), a member of the Potyvirus genus, is one of the significant viruses that infect peppers. This virus is transmitted in a non-persistent manner by aphids, posing a severe threat to the pepper industry in Zhejiang Province. To achieve rapid detection of this virus, this study established a detection method combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) with lateral flow diagnostic strips (LFD), enabling quick and visual detection of PepMoV in peppers. A set of RT-LAMP-specific primers was designed targeting the conserved sequences of the PepMoV coat protein (CP) gene. The optimized RT-LAMP-LFD method, under isothermal conditions at 63 ℃ for 40 minutes, followed by a 5-minute color reaction on the test strip, can complete the sample detection. This method demonstrates excellent performance in terms of specificity and sensitivity, achieving a 10-fold increase in sensitivity compared with traditional RT-qPCR. It provides an efficient and convenient tool for the rapid diagnosis of PepMoV, with potential for widespread application in pepper disease prevention and control.

Key words: pepper mottle virus, RT-LAMP-LFD, rapid detection, plant quarantine

中图分类号:

S436.3

付康, 张灿, 赵薇, 马宁, 俞晓平, 孙凯. 辣椒斑驳病毒RT-LAMP-LFD快速检测方法的建立[J]. 浙江农业学报, 2026, 38(2): 310-316.

FU Kang, ZHANG Can, ZHAO Wei, MA Ning, YU Xiaoping, SUN Kai. Establishment of a rapid detection method for pepper mottle virus using RT-LAMP-LFD[J]. Acta Agriculturae Zhejiangensis, 2026, 38(2): 310-316.

图/表 5

表1 RT-LAMP-LFD引物序列

Table 1 Primer sequences for RT-LAMP-LFD

引物名称 Primer name	引物序列(5'→3') Primer sequence(5'→3')	扩增片段长度/bp Amplification fragment length/bp	用途 Usage
F3	CTCATTTACTTGAATACAAACCAAG	29	RT-LAMP
B3	CTGTTCGTCTCCATCCAT	18	RT-LAMP
LB	GGTTTGGTGCATTGAAAATG	20	RT-LAMP
FIP(F1c+F2)¹	CCTCTTGTAGATCGTATGCTTTCATGTCGACATATCGAATACTCGT	46	RT-LAMP
BIP(B1c+B2)²	CAATGGGTACAGTGATGAATGGCCATGGTCCATGTTCCACTA	42	RT-LAMP
PepMoV-F	AGCAGCTCAAGATCAGAAACAT	22	RT-qPCR
PepMoV-R	TATATGCTTCAGCCACATCAG	21	RT-qPCR

图1 RT-LAMP引物在PepMoV外壳蛋白基因的位置分布箭头表示外引物(F3、B3)、前内引物FIP(F1c+F2)、后内引物BIP(B1c+B2)和环引物(LB)的位置和方向。

Fig.1 Distribution of RT-LAMP primers on the coat protein gene of PepMoV Arrows indicate the positions and orientations of the outer primers (F3, B3), the forward inner primer FIP (F1c+F2), the backward inner primer BIP (B1c+B2), and the loop primer (LB).

图2 RT-LAMP反应条件的优化上图为RT-LAMP产物的凝胶电泳图;下图为SYBR Green I染料可视化的RT-LAMP产物。a, MgSO4浓度;b, dNTPs浓度;c,反应时间;d,反应温度。M,GeneRuler 500 kb DNA梯度;-,阴性对照(无菌ddH2O作为模板)。

Fig.2 Optimization of the RT-LAMP reaction conditions The above figure is the gel electrophoresis diagram of RT-LAMP products, and the following figure shows the RT-LAMP products visualized with SYBR Green I dye. a, MgSO4 concentration; b, dNTPs concentration; c, Reaction time; d, Reaction temperature. M, GeneRuler 500 kb DNA ladder;-, Negative control (sterile ddH2O as template).

图3 RT-LAMP-LFD检测方法的特异性和灵敏度 a, RT-LAMP-LFD检测方法的建立。左上图为RT-LAMP产物的凝胶电泳图;左下图为SYBR Green I染料可视化的RT-LAMP产物;右图为RT-LAMP-LFD检测结果。M,GeneRuler 1 kb Plus DNA ladder;1,感染PepMoV的辣椒叶片样品;-,对照,健康辣椒叶片样品;C,控制线;T,测试线。b, RT-qPCR与RT-LAMP-LFD的灵敏度。上图为RT-qPCR产物的凝胶电泳图;中图为SYBR Green I染料可视化的RT-LAMP产物;下图为RT-LAMP-LFD检测结果。1~9,感染PepMoV的辣椒叶片总RNA的10倍梯度稀释(100~10-8);-,阴性对照(无菌ddH2O作为模板)。

Fig.3 Specificity and sensitivity of RT-LAMP-LFD detection method a, Establishment of RT-LAMP-LFD detection method. Upper left figure is gel electrophoresis of RT-LAMP products; Lower left figure is visualization of RT-LAMP products with SYBR Green I dye; Right figure is detection results of RT-LAMP-LFD. M, GeneRuler 1 kb Plus DNA ladder; 1, Pepper leaf samples infected with PepMoV;-, Control, healthy pepper leaf samples; C, Control line; T, Test line. b, Sensitivity of RT-qPCR and RT-LAMP-LFD. Top figure is gel electrophoresis of RT-qPCR products; Middle figure is visualization of RT-LAMP products with SYBR Green I dye; Bottom figure is RT-LAMP-LFD detection results. 1-9, The total RNA extracted from PepMoV-infected pepper leaves was diluted in a 10-fold gradient (100-10-8);-, Negative control (sterile ddH2O as template).

图4 基于RT-LAMP-LFD的田间辣椒样品检测上图为RT-qPCR产物的凝胶电泳图;下图为RT-LAMP-LFD检测结果。1~9,田间辣椒样品;-,阴性对照(无菌ddH2O作为模板)。

Fig.4 Detection of field pepper samples based on RT-LAMP-LFD Top figure is gel electrophoresis of RT-PCR products; Bottom figure is detection results of RT-LAMP-LFD. 1-9, Field pepper samples;-: Negative control (sterile ddH2O as template).

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辣椒斑驳病毒RT-LAMP-LFD快速检测方法的建立

Establishment of a rapid detection method for pepper mottle virus using RT-LAMP-LFD

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