[논문]Detection and Quantification of Apple Stem Grooving Virus in Micropropagated Apple Plantlets Using Reverse-Transcription Droplet Digital PCR

Kim, Sung-Woong; Lee, Hyo-Jeong; Cho, Kang Hee; Jeong, Rae-Dong

doi:10.5423/ppj.nt.05.2022.0072

[국내논문] Detection and Quantification of Apple Stem Grooving Virus in Micropropagated Apple Plantlets Using Reverse-Transcription Droplet Digital PCR 원문보기

The plant pathology journal, v.38 no.4, 2022년, pp.417 - 422

Kim, Sung-Woong (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) , Cho, Kang Hee (Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) , Jeong, Rae-Dong (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University)

Abstract ▼ AI-Helper

Apple stem grooving virus (ASGV) is a destructive viral pathogen of pome fruit trees that causes significant losses to fruit production worldwide. Obtaining ASGV-free propagation materials is essential to reduce economic losses, and accurate and sensitive detection methods to screen ASGV-free plantlets during in vitro propagation are urgently necessary. In this study, ASGV was sensitively and accurately quantified from in vitro propagated apple plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The optimized RT-ddPCR assay was specific to other apple viruses, and was at least 10-times more sensitive than RT-real-time quantitative PCR assay. Furthermore, the optimized RT-ddPCR assay was validated for the detection and quantification of ASGV using micropropagated apple plantlet samples. This RT-ddPCR assay can be utilized for the accurate quantitative detection of ASGV infection in ASGV-free certification programs, and can thus contribute to the production of ASGV-free apple trees.

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표/그림 (6)

표 Table 1. Detailed information for the primers and probe used in this study
그림 Fig. 1. Thermal gradient reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for optimizing annealing temperature for the detection of apple stem grooving virus. Eight RT-ddPCR reactions (with an annealing temperature gradient ranging from 48°C to 62°C) were performed to optimize the annealing temperature. The horizontal pink line represents the threshold; positive droplets (blue) and negative droplets (gray) are shown above and below the line, respectively. The X and Y axes represent the number of droplets and fluorescence signal amplitude, respectively.
그림 Fig. 2. Specificity of reverse transcription droplet digital polymerase chain reaction (assay for apple stem grooving virus (ASGV). Each sample is separated by vertical yellow lines and represents the fluorescence amplitude of apple chlorosis leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple skin scar viroid (ASSVd), and the negative template control (NTC).
표 Table 2. Sensitivity of the RT-qPCR and RT-ddPCR assay for ASGV
표 Table 3. Concentration of ASGV in micropropagated apple plantlet samples by RT-PCR, RT-qPCR, and RT-ddPCR
그림 Fig. 3. Detection and absolute quantification of apple stem grooving virus in micropropagated apple plantlets by reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Lanes 1-3; non-template control (NTC), lanes 4-40; RNA samples from selected propagated apple plantlets. RT-ddPCR amplitude plot showing droplets with distinguished positives (mostly between 3,000 and 6,000) and negatives (mostly below 2,000) in 37 apple plantlet samples.

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