Rapid and Visual Detection of Barley Yellow Dwarf Virus by Reverse Transcription Recombinase Polymerase Amplification with Lateral Flow Strips

Abstract

Barley yellow dwarf virus (BYDV) has been a major viral pathogen causing significant losses of cereal crops including oats worldwide. It spreads naturally through aphids, and a rapid, specific, and reliable diagnostic method is imperative for disease monitoring and management. Here, we established a rapid and reliable method for isothermal reverse transcription recombinase polymerase amplification (RT-RPA) combined with a lateral flow strips (LFS) assay for the detection of BYDV-infected oat samples based on the conserved sequences of the BYDV coat protein gene. Specific primers and a probe for RT-RPA reacted and optimally incubated at 42 o C for 10 min, and the end-labeled amplification products were visualized on LFS within 10 min. The RT-RPA-LFS assay showed no cross-reactivity with other major cereal viruses, including barley mild mosaic virus, barley yellow mosaic virus, and rice black streaked dwarf virus, indicating high specificity of the assay. The sensitivity of the RT-RPA-LFS assay was similar to that of reverse transcription polymerase chain reaction, and it was successfully validated to detect BYDV in oat samples from six different regions and in individual aphids. These results confirm the out-standing potential of the RT-RPA-LFS assay for rapid detection of BYDV.

Application of Rapid and Reliable Detection of Cymbidium Mosaic Virus by Reverse Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Immunoassay

“…To date, there are a number of reports on the development of RPA-based assays for plant viruses ( Babu et al, 2018 ; Babujee et al, 2019 ; Kim et al, 2019 , 2020 , 2022 , 2016; Lu et al, 2018 ; Mekuria et al, 2014 ; Silva et al, 2014 ; Zhang et al, 2014 ) and viroids ( Hammond and Zhang, 2016 ; Ivanov et al, 2020 ; Kovalskaya and Hammond, 2022 ; Lee et al, 2020 ). The RT-RPA-LFI assays for detection of plant viruses and viroids were performed in accordance with the TwistDx kit and lateral flow strips were utilized as an end-point readout method ( Kim et al, 2022 ; Kovalskaya and Hammond, 2022 ). Most RPA assays were developed with a TwistAmp nfo kit which is required nfo probe as well as a specific primer set ( Babu et al, 2018 ; Ivanov et al, 2020 ; Kim et al, 2022 ).…”

“…The RT-RPA-LFI assays for detection of plant viruses and viroids were performed in accordance with the TwistDx kit and lateral flow strips were utilized as an end-point readout method ( Kim et al, 2022 ; Kovalskaya and Hammond, 2022 ). Most RPA assays were developed with a TwistAmp nfo kit which is required nfo probe as well as a specific primer set ( Babu et al, 2018 ; Ivanov et al, 2020 ; Kim et al, 2022 ). Here, we validated RPA reaction for the detection of CymMV with a TwistAmp basic kit without a probe.…”

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications

“…The results showed that the primer labeled RPA-LFS method could detect 10 3 copies of RNA within 30 min, and its half-maximal binding concentration was 22 times lower than the probe-dependent RPA-LFS ( Ivanov et al., 2021 ). RPA could also detect tomato apical stunt viroid ( Kovalskaya and Hammond, 2022 ) and barley yellow dwarf virus ( Kim et al., 2022 ) in plants. The application of RPA in virus detection often required reverse transcription first.…”

New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand

“…Like most PoU methodologies RPA reactions are isothermal and therefore do not require expensive equipment such as thermocyclers, but unlike LFIA they can reach PCR-comparable levels of sensitivity as demonstrated by the newly developed tomato yellow leaf curl virus and tomato spotted wilt virus assays [ 56 , 57 ]. A lateral flow readout has been added to RPA systems for plant virus detection, whereby labelled amplicons bind to membrane-immobilised complementary probes [ 57 , 58 , 59 ]. This adaptation has also been integrated into another commonly used isothermal detection, loop mediated amplification (LAMP), for the detection of both RNA and DNA plant viruses [ 60 , 61 , 62 ].…”