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

Kim, Na-Kyeong; Lee, Hyo-Jeong; Kim, Sang-Min; Jeong, Rae-Dong

doi:10.5423/ppj.nt.01.2022.0009

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

The plant pathology journal, v.38 no.2, 2022년, pp.159 - 166

Kim, Na-Kyeong (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University) , Lee, Hyo-Jeong (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University) , Kim, Sang-Min (Crop Foundation Research Division, National Institute of Crop Science, Rural Development Administration) , Jeong, Rae-Dong (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University)

Abstract ▼ AI-Helper

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℃ 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 outstanding potential of the RT-RPA-LFS assay for rapid detection of BYDV.

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그림 Fig. 1. Schematic representation of the RPA-LFS principle for the detection of BYDV. (A) Principle of the recombinase polymerase amplification. (B) Schematic diagram of the RPA-LFS. RPA-LFS, recombinase polymerase amplification combined with lateral flow strip; BYDV, barley yellow dwarf virus; FAM, 6-carboxyfluoreescein; THF, tetrahydrofuran.
그림 Fig. 2. Optimization of RT-RPA-LFS assay for BYDV detection. (A) Optimization of the reaction temperature of the assay. RT-RPALFS assays were performed on BYDV-infected oat leaves at 30, 33, 37, 42, and 46°C (lanes 1-5). Lane 6, healthy oat leaves. (B) Optimization of reaction time of the assay. RT-RPA-LFS assays were performed on BYDV-infected oat leaves for 1, 5, 10, 15, and 20 min (lanes 1-5). Lane 6, healthy oat leaves. The top line on the LFS is the control line, and the other line is the test line. RT-RPA-LFS, reverse transcription recombinase polymerase amplification combined with a lateral flow strips; BYDV, barley yellow dwarf virus.
그림 Fig. 3. Specificity and sensitivity of RT-RPA-LFS assay for detection of BYDV. (A) Lane 1, BYDV-infected sample (positive sample); lane 2, BaMMV and BaYMV co-infected sample; lane 3, RBSDV-infected sample; lane 4, healthy oat sample (negative sample); lane 5, no template control contained water. RT-PCR was performed to confirm the presence of tested viruses. RT-PCR products were visualized on 1.5% agarose gels. M, DNA marker; lane 1, BYDV-infected sample (positive sample); lane 2, BaMMV and BaYMV coinfected sample; lane 3, RBSDV-infected sample; lane 4, healthy oat sample (negative sample); lane 5, no template control contained water. Oat actin was used as an internal control. (B) Ten-fold serial dilutions of transcripts from BYDV CP were performed using RTRPA- LFS and RT-PCR. The RT-PCR products (~595-bp) were visualized on 1.5% agarose gels. Lanes 1-8, BYDV transcript 100 ng/ μl to 100 fg/μl. RT-RPA-LFS, reverse transcription recombinase polymerase amplification combined with a lateral flow strips; BYDV, barley yellow dwarf virus; BaMMV, barley mild mosaic virus; BaYMV, barley yellow mosaic virus; RBSDV, rice black streaked dwarf virus; RT-PCR, reverse transcription polymerase chain reaction; CP, coat protein.
그림 Fig. 4. Application of the RT-RPA-LFS assay for BYDV-infected oat leaves and detection of BYDV in individual aphids using the RTRPA- LFS assay. (A) RT-RPA-LFS and RT-PCR assays were performed to detect BYDV in oat samples collected from six different regions. Lanes 1-8, samples from Jeongeup; lanes 9-12, samples from Gangjin; lanes 13-15, samples from Haenam; lanes 16-18, samples from Iksan; lanes 19-20, samples from Yeoncheon. RT-PCR products (approximately 595 bp) for BYDV detection were visualized on 1.5% agarose gels. Oat actin level was used as an internal control. (B) BYDVs were detected in eight aphid samples using the RT-RPALFS assay. The same samples were positive in both the assays. RT-PCR products (approximately 595 bp) for BYDV detection were visualized on 1.5% agarose gels. An actin gene from Rhopalosiphum padi was used as an internal control (Kim et al., 2020). RT-RPALFS, reverse transcription recombinase polymerase amplification combined with a lateral flow strips; BYDV, barley yellow dwarf virus; RT-PCR, reverse transcription polymerase chain reaction.
표 Table 1. RT-RPA-LFS and RT-PCR assay for detection of BYDV from seven different regions
표 Table 1. Continued

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