Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Development of a Multiplex Reverse Transcription-Polymerase Chain Reaction Assay for the Simultaneous Detection of Three Viruses in Leguminous Plants

  • Park, Chung Youl (School of Applied Biosciences, Kyungpook National University) ;
  • Min, Hyun-Geun (School of Applied Biosciences, Kyungpook National University) ;
  • Lee, Hong-Kyu (School of Applied Biosciences, Kyungpook National University) ;
  • Maharjan, Rameswor (Crop Production Technology Research Division, NICS, RDA) ;
  • Yoon, Youngnam (Crop Production Technology Research Division, NICS, RDA) ;
  • Lee, Su-Heon (School of Applied Biosciences, Kyungpook National University)
  • Received : 2018.09.17
  • Accepted : 2018.11.02
  • Published : 2018.12.31
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Abstract

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed for the detection of Clover yellow vein virus (ClYVV), Peanut mottle virus (PeMoV), and Tomato spotted wilt virus (TSWV), which were recently reported to infect soybean and azuki bean in Korea. Species-specific primer sets were designed for the detection of each virus, and their specificity and sensitivity were tested using mixed primer sets. From among the designed primer sets, two combinations were selected and further evaluated to estimate the detection limits of uniplex, duplex, and multiplex RT-PCR. The multiplex RT-PCR assay could be a useful tool for the field survey of plant viruses and the rapid detection of ClYVV, PeMoV, and TSWV in leguminous plants.

Keywords

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Fig. 1. Simultaneous detection using the finally selected primer combinations for the three viruses (Clover yellow vein virus, ClYVV; Peanut mottle virus, PeMoV, and Tomato spotted wilt virus, TSWV) by multiplex PCR. Lane M: 1kb DNA ladder (BioFACT, Daejeon, Korea), Lane 1 and 8: 447 bp for ClYYV; Lane 2 and 9: 599 and 560 bp for PeMoV; Lane 3 and 10: 947 and 682 bp for TSWV; Lane 4 and 11: ClYVV+PeMoV; Lane 5 and 12: ClYVV+TSWV; Lane 6 and 13: PeMoV+TSWV; Lane 7 and 14: ClYVV+PeMoV+TSWV.

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Fig. 2. Sensitivity test of uniplex-, duplex- and multiplex-PCR for the three legume viruses. (A) uniplex-PCR test results of ClYYV, PeMoV, and TSWV using the first primer combination, (B) duplex-PCR test results of ClYVV+PeMoV, ClYVV+TSWV, and PeMoV+TSWV using the first combination, (C) multiplex-PCR test results of ClYVV+PeMoV+TSWV and comparison of the two different primer combination sets no. 1 (left) and 2 (right). Lane M: 1kb DNA ladder (BioFACT, Daejeon, Korea), Lane 1 to 8: 10-1 to 10-8 diluted cDNA.

Table 1. Oligonucleotide sequences for the two different primer combinations used for the detection of the three viruses

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References

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