Real-time loop-mediated isothermal amplification: an early-warning tool for quarantine plant pathogen detection

Area of Science:

• Plant Pathology
• Molecular Diagnostics
• Biosecurity

Background:

• Biological invasions of plant pathogens pose significant threats to agriculture and natural ecosystems.
• Early detection and rapid response frameworks are essential for mitigating invasion impacts, particularly at ports of entry.
• Current molecular detection methods, such as PCR, often require specialized laboratory equipment, limiting their use in field settings.

Purpose of the Study:

• To develop rapid, point-of-care diagnostic tools for key quarantine plant pathogens.
• To create loop-mediated isothermal amplification (LAMP) assays for detecting Xylella fastidiosa, Ceratocystis platani, and Phytophthora ramorum.
• To enable on-site detection of these pathogens, facilitating timely intervention and prevention of spread.

Main Methods:

• Development of three distinct loop-mediated isothermal amplification (LAMP) assays.
• Testing LAMP assays on DNA extracted from axenic cultures and infected plant tissues.
• Utilizing a portable instrument (Genie® II) for rapid isothermal amplification and detection.

Main Results:

• Successful development of LAMP assays for Xylella fastidiosa, Ceratocystis platani, and Phytophthora ramorum.
• Detection of target pathogen DNA within 30 minutes of isothermal amplification.
• High specificity and sensitivity achieved, with detection limits as low as 0.02 pg µL⁻¹ of DNA.

Conclusions:

• The developed LAMP assays provide a rapid, on-site detection method for critical invasive plant pathogens.
• These tools are highly suitable for use at ports of entry to intercept pathogens.
• The assays can also be valuable for monitoring and preventing the spread of invasive pathogens in natural ecosystems.