Establishment of the Recombinase Polymerase Amplification–Lateral Flow Dipstick Detection Technique for Fusarium oxysporum

Abstract

Fusarium oxysporum can cause crown rot, wilt, root rot, and many other important plant diseases worldwide. During the development of strawberry crown rot disease, it is transmitted from the mother plant to the strawberry seedling via the stolon with obvious characteristics of latent infection. Therefore, the rapid and timely detection of F. oxysporum can enable efficient disease management. In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection technique was developed for the rapid detection of F. oxysporum on strawberry targeting on the CYP51C gene, which is unique to Fusarium spp. This RPA-LFD detection technique was highly specific to F. oxysporum, other Fusarium and non-Fusarium fungi cannot be detected. The optimal reaction conditions for this detection were 39°C for 8 min. The limit of detection was 1 pg of F. oxysporum genomic DNA in a 50 µL reaction. A total of 46 strawberry plants with or without crown rot symptoms from Jiande, Changxing, and Haining in Zhejiang province were further assessed for infection of F. oxysporum using both RPA-LFD and traditional tissue isolation. The RPA-LFD test showed that 32 of the 46 strawberry plants were tested were positive, means 32 strawberry plants were infected with F. oxysporum. While F. oxysporum was isolated from 30 of the 46 tested strawberry plants These results suggest that our established RPA-LFD method can quickly, sensitively, and specifically detect F. oxysporum infection in strawberry.

Point-of-care potentials of lateral flow-based field screening for Mycoplasma bovis infections: a literature review

“…The working principle of the RPA-FLD involves amplification (RPA), detection (LFD), and visualization [ 61 , 62 ]. The uvrC and oppD-oppF genes were amplified from the genomic DNA extracted from M. bovis reference type strain PG45 using specialized forward and reverse primer pairs.…”

“…Generally, people consider RPA in conjunction with LFS to be a relatively simple and portable method, using the LF probe to avoid the challenges of multiplexing and non-specific amplification that RPA frequently faces. Yet, the technique still requires basic laboratory equipment, such as a heat block or water bath, for the amplification step [ 61 ]. The RPA reaction requires incubation at temperatures between 35°C and 42°C for 15-30 minutes [ 51 , 52 ].…”

Real-Time Monitoring on the Chinese Giant Salamander Using RPA-LFD

“…This technique allows for the amplification of the template to be completed efficiently within a brief timeframe, typically 20 to 30 min, and requires maintaining a consistent temperature, thus eliminating the need for thermocycling [21]. Furthermore, when RPA is combined with a lateral flow dipstick (LFD), it allows for rapid result visualization within minutes without complex procedures, making it ideal for field applications [22][23][24]. The utility of RPA technology spans various fields, owing to its fast detection speed, high sensitivity, and simplicity of operation [25][26][27][28].…”

A Lateral Flow-Recombinase Polymerase Amplification Method for Colletotrichum gloeosporioides Detection

“…This isothermal amplification technique offers several advantages, including field mobility, rapid results, and the ability to perform amplification directly on a sample material without the need for DNA extraction [23]. Furthermore, the combination of RPA with lateral flow (LF) assays allows for rapid detection and easy visualization of results, enhancing its practicality for on-site testing [24]. The application of this technology also reduces the cost requirements for specialist and complicated instruments.…”