Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Detection of Ralstonia solanacearum with Nested PCR and DNA Enzyme-Linked Immunosorbent Assay

Nested PCR과 DNA Enzyme-Linked Immunosorbent Assays를 이용한 Ralstonia solanacearum의 검출

  • 고영진 (다이아프로브㈜ 진단사업부) ;
  • 조홍범 (서경대학교 생물공학과)
  • Published : 2007.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

In this study, we used the method of guanidin isothiocyanate and boiling with Chelex-100 resin to extract genomic DNA of Ralstonia solanacearum from soil. It is more efficient than general protocols to remove inhibitory compounds in soil and R. solanacearum on. Then, we applied polymerase chain reaction and DNA enzyme-linked immunosorbent assay (ELISA) to identify and detect pathogen. The fliC gene of R. solanacearum was selected for specific detection of pathogen and primer sets were designed. Among the primer sets, two specific and sensitive primer sets, RsolfliC(forward: 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGGCCTTCAGGGAGGTC-3, designed by J. $Sch\ddot{o}nfeld$ et al.) and RS_247 (forward: 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3 designed by this study), were designed to perform nested PCR. Nested PCR primer was labeled with biotin for hybridization between nested PCR product and probe to analyze with DNA ELISA.

본 연구는 polymerase chanin reaction(PCR)기법과 DNA enzyme-linked immunosorbent assay(DNA ELISA) 기법을 이용하여 토양내 식물병원균인 Ralstonia solanacearum를 검출하고자 하였다. 토양 시료로부터 분석에 사용될 R. solanacearum DNA를 추출하기 위하여 몇 가지 방법을 비교 평가한 결과 기존의 DNA 추출 방법에 비하여 Guanidin isothiocyanate와 Chelex-100 resin을 사용하는 방법 이 토양 내에 존재하는 다양한 중류의 반응 저해 물질과 R. solanacearum만의 고유한 PCR반응 저해물질들을 제거하는 데에 효과적이었다. R. solanacearum만을 특이적으로 검출하기 위해 fliC유전자 부위에 특이적인 몇 종의 primer들을 제작하였다. 이들 중 높은 민감도와 특이도를 나타내는 두 set의 primer RsolfliC(forward; 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGGCCTTCAGGGAGGTC-3, designed by J. $Sch\ddot{o}nfeld$ et al.)와 RS_247 (forward; 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3, designed by this study)를 선정하여 nested PCR을 수행할 수 있도록 고안하였다. Nested PCR primer에 biotin을 표지하였고 nested PCR산물의 내부 서열과 특이적으로 교잡반응을 할 수 있는 probe를 제작하여 PCR 결과를 DNA-EIA반응으로 확인 분석할 수 있도록 하였다. Primary PCR과 nested PCR의 산물을 전기영동 상에서 확인한 결과, nested PCR이 약 $10^2$정도의 높은 민감도를 나타내었고 DNA-EIA의 경우 $10^2P{\sim}10^3$정도의 민감도를 상승시켜주는 것으로 확인되었다.

Keywords

References

  1. 농촌진흥청 농업과학기술원. 2000. 채소 병해충 진단과 방제
  2. Boudazin, G., A.C. Le Roux, K. Josi, P. Labarre, and B. Jouan. 1999. Design of division specific primers of Ralstonia solanacearum and application to the identification of European isolates. Eur. J. Plant Pathol. 105,373-380 https://doi.org/10.1023/A:1008763111230
  3. Brian, E.G. and R.S. Todd. 2001. The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection. Appl. Environ. Microbiol. 67, 3866-3872 https://doi.org/10.1128/AEM.67.9.3866-3872.2001
  4. Buck, G.E. 1996. Detection of Bordetella pertussis by rapid-cycle PCR and colorimetric microwell hybridization. J Clin. Microbiol. 34, 1355-1358
  5. Chevrier, D., M.Y. Popoff, M.P. Dion, and D. Hermant. 1995 Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate. FEMS Immunol. Med. Microbiol. 10, 245-252 https://doi.org/10.1099/00222615-10-2-245
  6. Cho, S.-N., G.M.E. van der Vliet, S. Park, S.-H. Baik, S.-K. Kim, Y. Chong, A.H.J. Kolk, P.R. Klatser, and J.-D. Kim. 1995. Colorimetric microwell plate hybridization assay for detection of amplified Mycobacterium tuberculosis DNA from sputum samples. J Clin. Microbiol. 33, 752-754
  7. Fegan, M., M. Taghavi, L.I. Sly, and A.C. Hayward. 1998. Phylogeny, diversity and molecular diagnostics of Ralstonia solanacearum, p. 19-33. In P. Prior, C. Allen, and J.G Elphinstone (ed.), Bacterial wilt disease-molecular and ecological aspects. SpringerVerlag, Berlin, Germany
  8. Fujita, S.-I., B.A. Lasker, T.J. Lott, E. Reiss, and C.J. Morrison. 1995. Microtitration plate enzyme immunoassay to detect PCRamplified DNA from Candida species in blood. J. Clin. Microbiol. 33, 962-967
  9. Hayward, H.C. 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu. Rev. Phytopathol. 29,65-87 https://doi.org/10.1146/annurev.py.29.090191.000433
  10. Holben, W.E. 1994. Isolation and purification of bacterial DNA from soil, p. 727-751. In R.W Weaver, S. Angle, P. Bottomley, D. Bezdicek, S. Smith, A. Tabatabai, and A. Wollum (ed.), Methods of soil analysis, part 2. Microbiological and biochemical properties. Soil Science Society of America, Inc., Madison, Wis., USA
  11. Jung, K.-J., B.-H. Kim, E. Kim, J.-S. So, and S.-C. Koh. 2002. Monitoring expression of bphC gene from Ralstonia eutropha H850 induced by plant terpenes in soil. Kor. J. Microbiol. 42, 340-343
  12. Katz, J.B., A.D. Alstad, G.A. Gustafson, and K.M. Moser. 1993. Sensitive identification of bluetongue virus serogroup by a colorimetric dual oligonucleotide sorbent assay of amplified viral nucleic acid. J. Clin. Microbiol. 31, 3028-3030
  13. Kelman, A. 1998. One hundred and one years of research on bacterial wilt, p. 1-5. In P. Prior, C. Allen, and J. Elphinstone (ed.), Bacterial wilt disease. Molecular and ecological aspects. SpringerVerlag, Heidelberg, Germany
  14. Lebech, A.-M., K. Hansen, F. Brandrup, O. Clemmensen, and L. Halkier-Sorensen. 2000. Diagnostic value of PCR for detection of Borrelia burgdorferi DNA in clinical specimens from patients with erythema migrans and Lyme neuroborreliosis. Mol. Diagn. 5, 139-150 https://doi.org/10.2165/00066982-200005020-00007
  15. Lee, Y.-A., S.-C. Fan, L.-Y. Chiu, and K.-C. Hsia. 2001. Isolation of an insertion sequence from Ralstonia solanacearum race 1 and its potential use for strain characterization and detection. Appl. Environ. Microbiol. 67, 3943-3950 https://doi.org/10.1128/AEM.67.9.3943-3950.2001
  16. Miller, D.N., J.E. Bryant, E.L. Madsen, and W.C. Ghiorse. 1999 Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl. Environ. Microbiol. 65,4715-4724
  17. Moissenet, D., P. Bidet, A. Garbarg-Chenon, G. Ariel. and H. VuThien. 2000. Ralstonia paucula (formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods. stud: of the 16S-23S spacer of the rRNA operon. and comparison with other Ralstonia Species (R. eutropha. R. pickcttii. R. gikmlii. and R. solanacearum). J. Clin. Microbiol. 39. 381-384 https://doi.org/10.1128/JCM.39.1.381-384.2001
  18. Pastrik, K.H. and E. Maiss. 2000. Detection of Rctlsronia .sokmacearum in potato tubers by polymerase chain reaction. J. plntopathol. 148, 619-626
  19. Poussier, S., P. Prior, J. Luisetti, C. Hayward. and M. Fegan. 2000. Partial sequencing of the hrpB and endoglucanase genes confirms and expands the known diversity within the Ralstonia solanacearum species complex. Syst. Appl. Microbiol. 23, 479-486 https://doi.org/10.1016/S0723-2020(00)80021-1
  20. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA
  21. Schfeld, J., H. Heuer, J.D. van Elsas, and K. Smalla, 2003. Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments. Appl. Environ. Microbiol. 69, 7248-7256 https://doi.org/10.1128/AEM.69.12.7248-7256.2003
  22. Seal, S.E., L.A. Jackson, J.P. Young, and M.J. Daniels. 1993. Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii, and the blood disease bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction. J Gen. Microbiol. 139, 1587-1594 https://doi.org/10.1099/00221287-139-7-1587
  23. Taghavi, M., C. Hayward, L.I. Sly, and M. Fegan. 1996. Analysis of the phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas syzygii, and the blood disease bacterium of banana based on 16S rRNA gene sequences. Int. J. Syst. Bacteriol. 46, 10-15 https://doi.org/10.1099/00207713-46-1-10
  24. Tanyuksel, M., C. Guney, E. Araz, M.A. Saracli, and L. Doganci. 2004. Performance of the immunoglobulin G avidity and enzyme immunoassay IgG/IgM screening tests for differentiation of the clinical spectrum of toxoplasmosis. Kor. J. Microbiol. 40, 211-215
  25. Van der Wolf, J.M., S.G.C. Vriend, P. Kastelein, E.H. Nijhuis, P.J. van Bekkum, and J.W.L. van Vuurde. 2000. Immunofluorescence colony-staining (IFC) for detection and quantification of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil and verification of positive results by PCR and dilution plating. Eur. J. Plant Pathol. 106, 123-133 https://doi.org/10.1023/A:1008728828144
  26. Van Elsas, J.D., P. Kastelein, P. van Bekkum, J.M. van der Wolf, P.M. de Vries, and L.S. van Overbeek. 2000. Survival of Ralstonia solanacearum biovar 2, the causative agent of potato brown rot, in field and microcosm soils in temperate climates. Phytopathology 90, 1358-1366 https://doi.org/10.1094/PHYTO.2000.90.12.1358
  27. Vasse, J., P. Frey, and A. Trigalet. 1995. Microscopic studies of intercellular infection and protoxylem invasion of tomato roots by Pseudomonas solanacearum. Mol. Plant-Microbe Interact. 8, 241-251 https://doi.org/10.1094/MPMI-8-0241
  28. Wallis, F.M. and S.J. Truter. 1978. Histopathology oftomato plants infected with Pseudomonas solanacearum, with emphasis on ultrastructure. Physiol. Plant Pathol. 13, 307-317 https://doi.org/10.1016/0048-4059(78)90047-4
  29. Weller, S.A., J.G. Elphinstone, N.C. Smith, N. Boonham, and D.E. Stead. 2000. Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Appl. Environ. Microbiol. 66, 2853-2858 https://doi.org/10.1128/AEM.66.7.2853-2858.2000
  30. Winstanley, C. and J.A.W. Morgan. 1997. The bacterial flagellin gene as a biomarker for detection, population genetics and epidemiological analysis. Microbiology 143, 3071-3084 https://doi.org/10.1099/00221287-143-10-3071