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Implementation of an artificial reaction control in a TaqMan method for PCR detection of Ralstonia solanacearum race 3 biovar 2

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Abstract

A previously published TaqMan PCR test for R. solanacearum race 3 biovar 2 was modified to enable both the validation of negative results and the confirmation of positive results in a closed-tube system. Negative results were validated through the use of a reaction control plasmid, designated pRB2C2, which was designed to generate a 94bp product using the same amplimers targeting the primary diagnostic 68bp sequence in R. solanacearum race 3 biovar 2 DNA. SYBR Green was included in the reaction mix to facilitate the identification of post-reaction products using melt peak analysis. The 94bp reaction control had a melt peak temperature of about 90°C, while the diagnostic target amplicon had a melt peak temperature of about 83°C; thus positive results could be easily confirmed and distinguished from the reaction control product. Addition of pRB2C2 at 100 copies per reaction had no effect on the sensitivity of the TaqMan assay for R. solanacearum race 3 biovar 2, and the modified assay successfully detected R. solanacearum race 3 biovar 2 in infected, asymptomatic tomato stems and leaves as well as in potato tubers and stems.

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Abbreviations

PCR:

polymerase chain reaction

RFLP:

restriction fragment length polymorphism

CFBP:

collection française de bactéries phytopathogènes

References

  • Bain, R. A., & Pérombelon, C. A. (1988). Methods of testing potato cultivars for resistance to soft rot of tubers caused by Erwinia carotovora subsp. atroseptica. Plant Pathology, 37, 431–437. doi:10.1111/j.1365-3059.1988.tb02096.x.

    Google Scholar 

  • Bickley, J., & Hopkins, D. (1999). Inhibitors and Enhancers of PCR. In G. C. Saunders, & H. C. Parkes (Eds.), Analytical Molecular Biology: Quality and Validation (pp. 81–102). Cambridge: Thomas Graham House.

    Google Scholar 

  • Boudazin, G., Le Roux, A. C., Josi, K., Labarre, P., & Jouan, B. (1999). Design of division specific primers of Ralstonia solanacearum and application to the identification of European isolates. European Journal of Plant Pathology, 105, 373–380. doi:10.1023/A:1008763111230.

    Article  CAS  Google Scholar 

  • De Boer, S. H., Ward, L. J., Li, X., & Chittaranjan, S. (1995). Attenuation of PCR inhibition in the presence of plant compounds by addition of BLOTTO. Nucleic Acids Research, 23, 2567–2568. doi:10.1093/nar/23.13.2567.

    Article  PubMed  Google Scholar 

  • Fegan, M., Holoway, G., Hayward, A. C., & Timmis, J. (1998). Development of a diagnostic test based on the polymerase chain reaction (PCR) to identify strains of R. solanacearum exhibiting the biovar 2 genotype. In P. Prior, C. Allen, & J. Elphinstone (Eds.), Bacterial Wilt Disease, Molecular and Ecological Aspects (pp. 34–43). Berlin: Springer-Verlag.

    Google Scholar 

  • Glick, D. L., Coffey, C. M., & Sulzinski, M. A. (2002). Simultaneous PCR detection of the two major bacterial pathogens of geranium. Journal of Phytopathology, 150, 54–59. doi:10.1046/j.1439-0434.2002.00716.x.

    Article  CAS  Google Scholar 

  • Grousset, F., Roy, A. -S., & Smith, I. M. (1998). Situation of Ralstonia solanacearum in the EPPO region in 1997. Bulletin OEPP. EPPO Bulletin. European and Mediterranean Plant Protection Organisation, 28, 53–63. doi:10.1111/j.1365-2338.1998.tb00702.x.

    Google Scholar 

  • Janse, J. D. (1996). Potato brown rot in western Europe—history, present occurrence and some remarks on possible origin, epidemiology and control strategies. Bulletin OEPP. EPPO Bulletin. European and Mediterranean Plant Protection Organisation, 26, 679–695. doi:10.1111/j.1365-2338.1996.tb01512.x.

    Google Scholar 

  • Janse, J. D., & Schans, J. (1998). Experiences with the diagnosis and epidemiology of bacterial brown rot (Ralstonia solanacearum) in The Netherlands. Bulletin OEPP. EPPO Bulletin. European and Mediterranean Plant Protection Organisation, 28, 65–67. doi:10.1111/j.1365-2338.1998.tb00703.x.

    Google Scholar 

  • Ji, P., Allen, C., Sanchez-Perez, A., Yao, J., Elphinstone, J. G., Jones, J. B., et al. (2007). New diversity of Ralstonia solanacearum strains associated with vegetable and ornamental crops in Florida. Plant Disease, 91, 195–203. doi:10.1094/PDIS-91-2-0195.

    Article  CAS  Google Scholar 

  • Pastrik, K. -H., Elphinstone, J. G., & Pukall, R. (2000). Sequence analysis and detection of Ralstonia solanacearum by multiplex PCR amplification of 16S–23S ribosomal intergenic spacer region with internal positive control. European Journal of Plant Pathology, 108, 831–842. doi:10.1023/A:1021218201771.

    Article  Google Scholar 

  • Poussier, S., & Luisetti, J. (2000). Specific detection of biovars of Ralstonia solanacearum in plant tissues by nested-PCR-RFLP. European Journal of Plant Pathology, 106, 255–265. doi:10.1023/A:1008742609761.

    Article  CAS  Google Scholar 

  • Prior, P., & Fegan, M. (2005). Recent developments in the phylogeny and classification of Ralstonia solanacearum. Acta Horticulturae, 695, 127–136 ISHS.

    CAS  Google Scholar 

  • Seal, S. E., Jackson, L. A., Young, J. P. W., & Daniels, M. J. (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. Journal of General Microbiology, 139, 1587–1594.

    PubMed  CAS  Google Scholar 

  • Schönfeld, J., Heuer, H., van Elsas, J. D., & Smalla, K. (2003). Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments. Applied and Environmental Microbiology, 69, 7248–7256. doi:10.1128/AEM.69.12.7248-7256.2003.

    Article  PubMed  Google Scholar 

  • Siebert, P. D., & Larrick, J. W. (1992). Competitive PCR. Nature, 359, 557–558. doi:10.1038/359557a0.

    Article  PubMed  CAS  Google Scholar 

  • Smith, D. S., De Boer, S. H., & Gourley, J. (2008). An internal reaction control for routine detection of Clavibacter michiganensis subsp. sepedonicus using a real-time Taqman PCR-based assay. Plant Disease, 92, 684–693. doi:10.1094/PDIS-92-5-0684.

    Article  CAS  Google Scholar 

  • Tans-Kersten, J., Guan, Y., & Allen, C. (1998). Ralstonia solanacearum pectin methylesterase is required for growth on methylated pectin but not for bacterial wilt virulence. Applied and Environmental Microbiology, 64, 4918–4923.

    PubMed  CAS  Google Scholar 

  • Weller, S. A., Elphinstone, J. G., Smith, N. C., Boonham, N., & Stead, D. E. (2000). Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (Taqman) assay. Applied and Environmental Microbiology, 66, 2853–2858. doi:10.1128/AEM.66.7.2853-2858.2000.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Charlottetown Laboratory, Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada

    Donna S. Smith & Solke H. De Boer

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  1. Donna S. Smith
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  2. Solke H. De Boer
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Correspondence to Donna S. Smith.

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Smith, D.S., De Boer, S.H. Implementation of an artificial reaction control in a TaqMan method for PCR detection of Ralstonia solanacearum race 3 biovar 2. Eur J Plant Pathol 124, 405–412 (2009). https://doi.org/10.1007/s10658-008-9427-6

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  • DOI: https://doi.org/10.1007/s10658-008-9427-6

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