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Rapid Detection of Fusarium oxysporum Using Insulated Isothermal PCR and a Rapid, Simple DNA Preparation Protocol
Tsai-De Chang1, Li-Nian Huang1, Yi-Jia Lin1
1Department of Plant Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
Abstract:
We developed an insulated isothermal PCR (iiPCR) method for the efficient and rapid detection of Fusarium oxysporum (Fo), which is a fungus that infects various hosts and causes severe crop losses. The Fo iiPCR method was sensitive enough to detect up to 100 copies of standard DNA template and 10 fg of Fo genomic DNA. In addition, it could directly detect 1 pg of mycelium and 10 spores of Fo without DNA extraction. Our study compared the performance of Fo iiPCR to that of three published in planta molecular detection methods-conventional PCR, SYBR green-based real-time PCR, and hydrolysis probe-based real-time PCR-in field detection of Fo. All diseased field samples yielded positive detection results with high reproducibility when subjected to an Fo iiPCR test combined with a rapid DNA extraction protocol compared to Fo iiPCR with an automated magnetic bead-based DNA extraction protocol. Intraday and interday assays were performed to ensure the stability of this new rapid detection method. The results of detection of Fo in diseased banana pseudostem samples demonstrated that this new rapid detection method was suitable for field diagnosis of Fusarium wilt and had high F1 scores for detection (the harmonic mean of precision and recall of detection) for all asymptomatic and symptomatic Fo-infected banana samples. In addition, banana samples at four growth stages (seedling, vegetative, flowering and fruiting, and harvesting) with mild symptoms also showed positive detection results. These results indicate that this new rapid detection method is a potentially efficient procedure for on-site detection of Fo.
Insights
We developed a rapid insulated isothermal PCR (iiPCR) method for detecting Fusarium oxysporum (Fo). This sensitive technique allows for direct detection of fungal DNA, mycelium, and spores, aiding in quick crop disease diagnosis.
Area of Science:
- Plant Pathology
- Molecular Biology
- Biotechnology
Background:
- Fusarium oxysporum (Fo) causes significant crop losses globally.
- Accurate and rapid detection of Fo is crucial for effective disease management.
- Existing molecular detection methods can be time-consuming and require extensive sample preparation.
Purpose of the Study:
- To develop and validate a novel insulated isothermal PCR (iiPCR) method for rapid and sensitive detection of Fusarium oxysporum.
- To compare the performance of the new iiPCR method against established in planta molecular detection techniques.
- To assess the suitability of the iiPCR method for on-site field diagnosis of Fusarium wilt in various crop stages.
Main Methods:
- Development of an insulated isothermal PCR (iiPCR) assay for Fusarium oxysporum detection.
- Evaluation of the assay's sensitivity using standard DNA templates, genomic DNA, mycelium, and spores.
- Comparative analysis of iiPCR with conventional PCR, SYBR green real-time PCR, and hydrolysis probe real-time PCR using field samples.
- Assessment of reproducibility, stability (intraday and interday assays), and F1 scores for detection in banana pseudostem samples.
Main Results:
- The Fo iiPCR method demonstrated high sensitivity, detecting down to 100 DNA copies, 10 fg genomic DNA, 1 pg mycelium, and 10 spores without DNA extraction.
- The iiPCR method, especially with a rapid DNA extraction protocol, showed high reproducibility and positive detection in diseased field samples.
- Comparative studies indicated the iiPCR method's suitability for field diagnosis, achieving high F1 scores for both symptomatic and asymptomatic infected banana samples across different growth stages.
Conclusions:
- The developed insulated isothermal PCR (iiPCR) method offers an efficient, rapid, and sensitive approach for detecting Fusarium oxysporum.
- The iiPCR method is suitable for direct detection of fungal components and can be effectively used for on-site field diagnosis of Fusarium wilt.
- This technology holds potential for improving disease management strategies through timely and accurate pathogen identification.
