Establishment of RT-RPA-LFD Method for Rapid Detection of Telosma Mosaic Virus in Passiflora

doi:10.16420/j.issn.0513-353x.2023-0701

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (11): 2701-2709.doi: 10.16420/j.issn.0513-353x.2023-0701

• New Technology and New Method • Previous Articles Next Articles

Establishment of RT-RPA-LFD Method for Rapid Detection of Telosma Mosaic Virus in Passiflora

DING Huijiao¹^,², WANG Hao¹, BAO Fangwei¹, SUN Geng¹, PANG Jingjun¹, FAN Zaifeng¹^,², ZHAO Wenjun³^,^*(), ZHOU Tao¹^,²^,^*()

¹ College of Plant Protection，China Agricultural University，Beijing 100193，China
² Sanya Institute，China Agricultural University，Sanya，Hainan 572025，China
³ Center for Biosafety，Chinese Academy of Inspection and Quarantine，Sanya，Hainan 572019，China

Received:2024-05-27 Revised:2024-08-29 Online:2024-12-12 Published:2024-11-26
Contact: ZHAO Wenjun, ZHOU Tao

Abstract

Abstract:

Primers and probe were designed according to the coat protein（CP）gene of telosma mosaic virus（TeMV）on passion fruit，and recombinase polymerase amplification（RPA）was combined with lateral flow dipstick（LFD）to establish a method for rapid detection of TeMV. The reaction time，temperature，probe concentration and primers concentration of the method were optimized，and then the specificity and sensitivity of the metho also were examined. The experimental results show that the optimal reaction temperature was 37 ℃，optimal reaction time was 13 min，optimal probe concentration was 5 μmol · L^-1，and the optimal primers concentration was 10 μmol · L^-1. The method can specifically detect TeMV，and the minimum detection limit could reach to 5.125 × 10^-4 ng · μL^-1 for plasmids containing TeMV cp，which is ten thousand times more sensitive than ordinary PCR detection. This method has high sensitivity and specificity，which can be used to detect passion fruit samples in the field.

Key words: Passiflora, telosma mosaic virus, recombinase polymerase amplification, lateral flow dipstick, detection, visualization

DING Huijiao, WANG Hao, BAO Fangwei, SUN Geng, PANG Jingjun, FAN Zaifeng, ZHAO Wenjun, ZHOU Tao. Establishment of RT-RPA-LFD Method for Rapid Detection of Telosma Mosaic Virus in Passiflora[J]. Acta Horticulturae Sinica, 2024, 51(11): 2701-2709.

Figures/Tables 6

Fig. 1 Binding sites of RPA primers in the cp gene sequence of TeMV Italics indicate RPA primers.

Fig. 2 A leaf of Passiflora edulis with mosaic symptoms

Fig. 3 Optimization of reaction conditions for RT-RPA-LFD detection system C：Control line；T：Test line. The same below.

Fig. 4 RT-PCR detection and RT-RPA-LFD detection of TeMV，SCMV，TuMV，PPV，PVMV，CABYV and PLV M：DNA marker. The target bands of TeMV detected by RT-PCR were 398 bp and 816 bp，respectively.

Fig. 5 Detection of different concentrations TeMV cp-containing plasmids by ordinary RT-PCR and RT-RPA-LFD

Fig. 6 Passion fruit field samples were detected for TeMV by RT-RPA-LFD and RT-PCR +：Positive control；-：Negative control；1-14：Passion fruit samples.

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Establishment of RT-RPA-LFD Method for Rapid Detection of Telosma Mosaic Virus in Passiflora

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