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Development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Pseudomonas syringae pv. tomato in planta

  • Zhen-Dong Chen
  • Hua-Jun Kang
  • A-Li ChaiEmail author
  • Yan-Xia Shi
  • Xue-Wen Xie
  • Lei Li
  • Bao-Ju LiEmail author
Article
  • 15 Downloads

Abstract

Tomato bacterial speck, caused by Pseudomonas syringae pv. tomato (Pst), is one of the most devastating bacterial diseases in tomato worldwide. To establish a rapid amplification system for the detection of Pst, a loop-mediated isothermal amplification (LAMP) method, which includes two external primers (F3/B3), two internal primers (FIP/BIP) and one backward loop primer (B-Loop), was designed based on the hrpZ gene. The specificity of the LAMP primer set was widely validated on Pst and non-target strains. The conditions for LAMP detection of Pst were optimized to complete in 60 min at 63 °C. The amplification was confirmed through gel electrophoresis or visual inspection using calcein. In the sensitivity test, the detection limit of the LAMP assay was 1.61 × 10 fg μL−1 for genomic DNA and 1.05 × 103 CFU mL−1 for bacterial suspension without DNA extraction. The novel method was also applied for the detection of Pst in artificially and naturally infected tomato leaf and stem tissue samples, and even the early onset of disease could be detected by the assay. This method can rapidly detect Pst-infected tissues without strain enrichment and complex DNA extraction, and hence, it is suitable for quarantine and field detection.

Keywords

Tomato bacterial speck Pseudomonas syringae pv. tomato Loop-mediated isothermal amplification (LAMP) Visualization 

Notes

Acknowledgements

This work was supported by the National key research & development (R&D) plan (No. 2017YFD0200300), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (No. CAAS-ASTIP-IVFCAAS), and Modern Agro-industry Technology Research System in China (No. CARS-25).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study does not contain any studies with human participants or animals performed by any of the authors.

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Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2020

Authors and Affiliations

  1. 1.Institute of Vegetables and FlowersChinese Academy of Agricultural SciencesBeijingChina
  2. 2.Vegetable Research InstituteGuangxi Academy of Agricultural SciencesNanningChina

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