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

doi:10.3969/j.issn.1004-1524.20240711

Acta Agriculturae Zhejiangensis ›› 2026, Vol. 38 ›› Issue (2): 310-316.DOI: 10.3969/j.issn.1004-1524.20240711

• Plant Protection • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S436.3

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.

Figures/Tables 5

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

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).

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).

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).

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

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