DOI QR코드

DOI QR Code

Improvement of RT-PCR Sensitivity for Fruit Tree Viruses by Small-scale dsRNA Extraction and Sodium Sulfite

  • Lee, Sin-Ho (National Horticultural Research Institute, Rural Development Administration) ;
  • Kim, Hyun-Ran (National Horticultural Research Institute, Rural Development Administratio) ;
  • Kim, Jae-Hyun (National Horticultural Research Institute, Rural Development Administratio) ;
  • Kim, Jeong-Soo (National Horticultural Research Institute, Rural Development Administration)
  • 발행 : 2004.06.01

초록

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

키워드

참고문헌

  1. Baek, K. H. 2002. Development of diagnostic methods to identify the major fruit viruses. Ministry of Agriculture and Forestry. 73
  2. Brunt, A. A., Crabtree, K., Dallwitz, M. J., Gibbs, A. J. and Watson, L. 1996. Viruses of plants, Descriptions and Lists from the VICE Database. CAB International
  3. Campbell, A. I. 1963. The effect of some latent virus infections on the growth and cropping of apples. J. Hort. Sci. 38: 15- I9
  4. Demeke, T. and Adams, R. P, 1992. The effects of plant polysaccharides and buffer additives on PCR. Biotechlliques 12:332-334
  5. Desvignes,J.-C. 1999. Virus diseases of fruit trees. Ctifl, Paris
  6. Henson, J. and French, R. 1993. The polymerase chain reaction and plant disease diagnosis. Annu. Rev. Phytopathol. 143:369-373
  7. John, M. E. 1992. An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Res. 20:2381 https://doi.org/10.1093/nar/20.9.2381
  8. Kim, H. R., Chung, J. D., Chung, B. N., Lee, B. C., Park, J. W. and Choi, Y. M. 2001. Improved detection and purification of Grapevine leafroll-associated 3 Closterovirus using tissue culture. Korean J. Plant TIssue Culture 28:335-339
  9. Mackenzie, D. J., Mclean, M. A., MurkeIji, S. and Green, M. 1997. Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant Dis. 81:222-226 https://doi.org/10.1094/PDIS.1997.81.2.222
  10. Matthews, R. E. F. 1991. Plant Virology, $3^{rd}$ edition. Academic Press, New York
  11. Molnar-Perl, I. and Friedman, M. 1990. Inhibition of browning by sulfur amino acids. 3. Apples and potatoes. J. Agric. Food Chern. 38:1652-1656 https://doi.org/10.1021/jf00098a006
  12. Monette, P. L. and James, D. 1990. Detection of two strains of Grapevine virus A. Plant Dis. 74:898-900 https://doi.org/10.1094/PD-74-0898
  13. Morris, T. J. and Dodds, J. A. 1979. Isolation and analysis of double- stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69:854-858 https://doi.org/10.1094/Phyto-69-854
  14. Nakahara, K., Hataya, T. and Uyeda, I. 1999. A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR. J. Virol. Methods 77:47-58 https://doi.org/10.1016/S0166-0934(98)00135-9
  15. Nassuth, A., Pollari, E., Helmeczy, K., Stewart, S. and Kofalvi, S. A. 2000. Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts. J. Virol. Methods 90:37-49 https://doi.org/10.1016/S0166-0934(00)00211-1
  16. Newbury, H. J. and Possingham, J. V. 1977. Factors affecting the extraction of intact ribonucleic acid from plant tissue containing interfering phenolic compounds. Plant Physiol. 60:543-547 https://doi.org/10.1104/pp.60.4.543
  17. Okuda, M. and Hanada, K. 2001. RT-PCR for detecting five distinct tospovirus species using degenerate primers and dsRNA template. J. Virol. Methods 96: 149-156 https://doi.org/10.1016/S0166-0934(01)00321-4
  18. Pandey, R. N., Adams, R. P. and Flournoy, L. E. 1996. Inhibition of random amplified polymorphic DNAs (RAPDs) by plant polysaccharides. Plant Mol. BioI. Reporter 14:17-22 https://doi.org/10.1007/BF02671898
  19. Posnette, A. F., Cropley, R. and Ellenberger, C. E. 1963. The effect of virus infection on the growth and crop of apple, pear, and plum trees. Phytopathol. Mediterranea 2:158-161
  20. Rezaian, M. A. and Krake, L. R. 1987. Nucleic acid extraction and virus detection in grapevine. J. Virol. Methods 17:277-285 https://doi.org/10.1016/0166-0934(87)90137-6
  21. Rowhani, A., Chay, C., Golino, D. A. and Falk, B. W. 1993. Development of a polymerase chain reaction technique for the detection of grapevine fanleaf virus in grapevine tissue. Phytopathology 83:749-753 https://doi.org/10.1094/Phyto-83-749
  22. Staub, U., Polivca, H. and Gross, H. J. 1995. Two rapid microscale procedures for isolation of total RNA from leaves in polyphenols and polysaccharides: application for sensitive detection of grapevine viroids. J. Virol. Methods 52:209-218 https://doi.org/10.1016/0166-0934(94)00165-D
  23. Wu, H. C., Chu, H. L., Kuo, J. M., Huang, L. C. and Shaw, J. F. 1999. The biochemical characteristics of polyphenol oxidase from browning tissue-cultured bamboo (Dendrocalarnus latifloTUs). Food Sci. Agric. Chem. 1:244-249
  24. Zaitlin, M. and Hull, R. 1987. Plant virus-host interactions. Annu. Rev. Plnat Physiol. 38:291-315 https://doi.org/10.1146/annurev.pp.38.060187.001451
  25. Zhang, Y.-P. and Rowhani, A. 2000. A strategy for rapid cDNA cloning from double-stranded RNA templates isolated from plant infected with RNA viruses by using Taq DNA polymerase. J. Virol. Methods 84:59-63 https://doi.org/10.1016/S0166-0934(99)00126-3
  26. Zhang, Y.-P., Uyemoto, J. K. and Kirkpatriek, B. C. 1998. A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay. J. Virol. Methods 71 :45-50 https://doi.org/10.1016/S0166-0934(97)00190-0

피인용 문헌

  1. Complete genome sequence of narcissus late season yellows virus infecting Chinese narcissus in China vol.157, pp.9, 2012, https://doi.org/10.1007/s00705-012-1328-5
  2. Occurrence of Stone Fruit Viruses on Peach Trees (Prunus persica L. Batsch) in Korea vol.18, pp.4, 2012, https://doi.org/10.5423/RPD.2012.18.4.391