Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Improvement of Antigen Blotting in a Tissue Blot Immunobinding Assay for the Detection of Two Chili Pepper Viruses

  • Han, Jung-Heon (Center for Plant Molecular Genetics and Breeding Research, Seoul National University) ;
  • Shin, Jun-Sung (Center for Plant Molecular Genetics and Breeding Research, Seoul National University) ;
  • Kim, Young-Ho (Department of Agricultural Biotechnology and Center for Plant Molecular Genetics and Breeding Research, Seoul National University) ;
  • Kim, Byung-Dong (Department of Plant Science and Center for Plant Molecular Genetics and Breeding Research)
  • Published : 2007.11.30
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Abstract

The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ring-blot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of R-BIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the R-BIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as whole-leaf blotting and crushed-leaf blotting), suggesting that the R-BIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities.

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References

  1. Abad, J. A. and J. W. Moyer. 1992. Detection of sweet potato feathery mottle virus by in vitro transcribed RNA probes and serological assays. Phytopathology 82: 300-305 https://doi.org/10.1094/Phyto-82-300
  2. Chia, T.-F., Y.-S. Chan, and N.-H. Chua. 1992. Detection and localization of viruses in orchids by tissue-print hybridization. Plant Pathol. 41: 355-361 https://doi.org/10.1111/j.1365-3059.1992.tb02358.x
  3. Clark, M. F. 1981. Immunosorbent assays in plant pathology. Annu. Rev. Phytopathol. 19: 83-106 https://doi.org/10.1146/annurev.py.19.090181.000503
  4. Colinet, D., J. Kummert, P. Lepoivre, and J. Semal. 1994. Identification of distinct potyviruses in mixedly-infected sweet potato by the polymerase chain reaction with degenerate primers. Phytopathology 84: 65-69 https://doi.org/10.1094/Phyto-84-65
  5. Han, J.-H., S.-H. Shon, and Y.-J. La. 2001. Identification and characterization of tobamoviruses isolated from commercial pepper seeds. Res. Plant Dis. 7: 164-169
  6. Han, J.-H., H.-S. Choi, D.-W. Kim, H.-R. Lee, and B.-D. Kim. 2006. Biological, physical and cytological properties of pepper mottle virus-SNU1 and its RT-PCR detection. Plant Pathol. J. 22: 155-160 https://doi.org/10.5423/PPJ.2006.22.2.155
  7. Hibi, T. and Y. Saito. 1985. A dot immunobinding assay for the detection of tobacco mosaic virus in infected tissues. J. Gen. Virol. 66: 1191-1194 https://doi.org/10.1099/0022-1317-66-5-1191
  8. Hsu, H. T., H. Hibino, and P. Q. Cabauatan. 1990. Development of serological procedures for rapid, sensitive, and reliable detection of rice grassy stunt virus. Plant Dis. 74: 695-698 https://doi.org/10.1094/PD-74-0695
  9. Jacobi, V., G. D. Bachand, R. C. Hamelin, and J. D. Castello. 1998. Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses. J. Virol. Meth. 74: 167-178 https://doi.org/10.1016/S0166-0934(98)00086-X
  10. Kim, H.-S., Y.-M. Bae, Y.-K. Kim, B.-K. Oh, and J.-W. Choi. 2006. Antibody layer fabrication for protein chip to detect E. coli O157:H7, using microcontact printing technique. J. Microbiol. Biotechnol. 16: 141-144
  11. Kim, P. I., B. D. Erickson, and C.E. Cerniclia. 2005. A membrane-array method to detect specific human intestinal bacteria in fecal samples using reverse transcriptase-PCR and chemiluminescence. J. Microbiol. Biotechnol. 15: 310-320
  12. La, Y.-J., J.-H. Han, G.-B. Sim, B.-D. Kim, and K.-K. Ahn. 1999. Detection of cymbidium mosaic virus and odontoglossum ringspot virus in orchid plants by tissue-blot immunoassay. J. Kor. Soc. Hort. Sci. 40: 481-484
  13. Lin, N. S., Y. H. Hsu, and H. T. Hsu. 1990. Immunological detection of plant viruses and a mycoplasma-like organism by direct tissue blotting on nitrocellulose membranes. Phytopathology 80: 824-828 https://doi.org/10.1094/Phyto-80-824
  14. Lommel, S. A., A. H. McCain, and T. J. Morris. 1982. Evaluation of indirect enzyme-linked immunosorbent assay for the detection of plant viruses. Phytopathology 72: 1018-1022 https://doi.org/10.1094/Phyto-72-1018
  15. Marco, S. and S. Cohen. 1979. Rapid detection and titer evaluation of viruses in pepper by enzyme-linked immunosorbent assay. Phytopathology 69: 1259-1262 https://doi.org/10.1094/Phyto-69-1259
  16. Park, K.-S., Y.-J. Bae, E.-J. Jung, and S.-J. Kang. 2005. RT-PCR-based detection of six garlic viruses and their phylogenetic relationships. J. Microbiol. Biotechnol. 15: 1110-1114
  17. Singh, R. P. 1998. Reverse-transcription polymerase chain reaction for the detection of viruses from plants and aphids. J. Virol. Meth. 74: 125-138 https://doi.org/10.1016/S0166-0934(98)00074-3
  18. Smith, O. P., V. D. Damsteegt, C. J. Keller, R. J. Beck, and A. D. Hewings. 1993. Detection of potato leafroll virus in leaf and aphid extracts by dot-blot hybridization. Plant Dis. 77: 1098-1102 https://doi.org/10.1094/PD-77-1098
  19. Somowiyarjo, S., N. Sako, and F. Nonaka. 1989. Dotimmunobinding assay for zucchini yellow mosaic virus using polyclonal and monoclonal antibodies. Ann. Phytopath. Soc. Japan 55: 56-63 https://doi.org/10.3186/jjphytopath.55.56
  20. Van Regenmortel, M. H. V. 1982. Serology and Immunochemistry of Plant Viruses. Academic Press, New York
  21. Varveri, C. 2000. Potato Y potyvirus detection by immunological and molecular techniques in plants and aphids. Phytoparasitica 28: 1-8
  22. Yoshikawa, N., P. Poolpol, and T. Inouye. 1986. Use of a dot immunobinding assay for rapid detection of strawberry pseudo mild yellow edge virus. Ann. Phytopath. Soc. Japan 52: 728-731 https://doi.org/10.3186/jjphytopath.52.728