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Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry
Wen Huang1, Hao Zhang2, Jingsheng Xu2
1Institute of Plant Protection, Chinese Academy of Agricultural SciencesBeijing, China; College of Plant Protection, Southwest UniversityChongqing, China.
Abstract:
Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 102 CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.
Insights
A new loop-mediated isothermal amplification (LAMP) method rapidly detects Ralstonia solanacearum phylotype I mulberry strains, the cause of mulberry bacterial wilt. This cost-effective diagnostic tool offers potential for accurate field disease forecasting.
Area of Science:
- Plant Pathology
- Microbiology
- Molecular Diagnostics
Background:
- Mulberry bacterial wilt is caused by Ralstonia solanacearum phylotype I strains.
- Existing diagnostic methods are not suitable for detecting mulberry bacterial wilt.
- Accurate and rapid diagnostics are crucial for effective disease management.
Purpose of the Study:
- To develop a rapid and sensitive diagnostic method for R. solanacearum phylotype I mulberry strains.
- To create a loop-mediated isothermal amplification (LAMP) assay for early detection of mulberry bacterial wilt.
Main Methods:
- Designed a set of six primers targeting the clone MG67 sequence for LAMP assay.
- Optimized reaction conditions to 64°C for 20 minutes.
- Evaluated specificity against 46 R. solanacearum strains and 7 other soil-borne bacteria; assessed sensitivity using genomic DNA and bacterial cells, including in the presence of plant tissue fluid.
Main Results:
- The developed LAMP assay detected R. solanacearum phylotype I mulberry strains within 20 minutes.
- High specificity was confirmed, with no cross-reactivity with other tested bacteria.
- The method demonstrated high sensitivity, detecting as little as 160 fg genomic DNA or 2.2 × 10^2 CFU/ml, unaffected by plant tissue fluid.
Conclusions:
- The developed LAMP assay is a rapid, specific, and sensitive tool for detecting R. solanacearum phylotype I mulberry strains.
- The assay's simplicity and low cost make it suitable for field applications.
- This diagnostic method has the potential to improve the accuracy and efficiency of mulberry bacterial wilt disease forecasting.
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