The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Rapid and Specific Detection of Apple stem grooving virus by Reverse Transcription-recombinase Polymerase Amplification

  • Kim, Nam-Yeon (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University) ;
  • Oh, Jonghee (School of Applied Biosciences, Kyungpook National University) ;
  • Lee, Su-Heon (School of Applied Biosciences, Kyungpook National University) ;
  • Kim, Hongsup (Seed Testing & Research Center, Korea Seed & Variety Service) ;
  • Moon, Jae Sun (Plant Genome Research Center, Korea Research Institute of Bioscience & Biotechnology) ;
  • Jeong, Rae-Dong (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University)
  • Received : 2018.06.18
  • Accepted : 2018.08.26
  • Published : 2018.12.01
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Abstract

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as $4.7ng/{\mu}l$ of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of $42^{\circ}C$. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

Keywords

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Fig. 1. Primer position within the coat protein of Apple stem grooving virus (ASGV) and the detection of ASGV by RT-RPA. (A) ClustalW multiple sequence alignment was performed with Bio-edit using the sequence of Korea ASGV isolates with isolates from other countries. Unfilled boxes represent the primer regions used in this study. (B) RT-RPA amplification products of ASGV. M, DNA marker; lane 1, ASGV-infected tissues; lane 2, non-infected tissues control. Five independent reactions were performed, and similar results were obtained.

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Fig. 2. Determination of optimal reaction time for ASGV diagnosis using RT-RPA. ASGV was amplified with RT-RPA for different time points and a clear DNA band of the expected size (143 bp) could be visualized by agarose gel electrophoresis. M, DNA marker; lanes 1-7, DNA products from reactions incubated for 1, 3, 5, 10, 15, 20, and 30 min, respectively

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Fig. 3. Sensitivity of RT-RPA. (A) The detection limit of the RTRPA assay using total RNA isolated from pear leaves infected with ASGV. M, DNA marker; lanes 1-5, serial 10-fold dilutions of RNA (ranging from 47 ng/μl to 4.7 pg/μl). (B) The detection limit of the RT-PCR assay using total RNA isolated from pear leaves infected with ASGV. M, DNA marker; lanes 1-5, serial 10-fold dilution of RNA (ranging from 47 ng/μl to 4.7 pg/μl).

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Fig. 4. Specificity of RT-RPA for ASGV. Total RNAs from the ASGV-, ACLSV-, and ASPV-infected tissues were all tested using RT-RPA. M, DNA marker; lane 1, ASGV-infected tissues; lane 2, ACLSV-infected tissues; lane 3, ASPV-infected tissues; lane 4, virus-free tissues; lane 5, no-template virus. Five independent reactions were performed with similar results observed for all five reactions.

Table 1. Primers used in this study

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