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

Abstract

An effective framework for early warning and rapid response is a crucial element to prevent or mitigate the impact of biological invasions of plant pathogens, especially at ports of entry. Molecular detection of pathogens by using PCR-based methods usually requires a well-equipped laboratory. Rapid detection tools that can be applied as point-of-care diagnostics are highly desirable, especially to intercept quarantine plant pathogens such as Xylella fastidiosa, Ceratocystis platani and Phytophthora ramorum, three of the most devastating pathogens of trees and ornamental plants in Europe and North America. To this aim, in this study we developed three different loop mediated isothermal amplification (LAMP) assays able to detect each target pathogen both in DNA extracted from axenic culture and in infected plant tissues. By using the portable instrument Genie^® II, the LAMP assay was able to recognize X. fastidiosa, C. platani and P. ramorum DNA within 30 min of isothermal amplification reaction, with high levels of specificity and sensitivity (up to 0.02 pg µL^-1 of DNA). These new LAMP-based tools, allowing an on-site rapid detection of pathogens, are especially suited for being used at ports of entry, but they can be also profitably used to monitor and prevent the possible spread of invasive pathogens in natural ecosystems.

Keywords: Alien pathogens; Canker Stain Disease; Isothermal amplification; LAMP; Olive Quick Decline Syndrome; Portable diagnostics; Sudden Oak Death.

Fig. 1

Selection of kinetics. Real time LAMP results reported as amplification and melting derivative plot for Xylella fastidiosa, Ceratocystis platani and Phytophthora ramorum including target species DNA (10 ng μL⁻¹) in red, COX endogenous plant gene (green) and symptomatic plant tissues (black continuous line). No template control (NTC) and healthy plant tissue were also included (black dotted line). T_a annealing temperature, t_amp amplification time

Fig. 2

Sensitivity results obtained by testing both on LAMP and qPCR 11-fold 1:5 serial dilution (ranged from 10 ng μL⁻¹ to 0.001 pg μL⁻¹) of each standard DNA template (X. fastidiosa—Co.Di.Ro strain; C. platani—isolate Cp24; P. ramorum- isolate Pram). LAMP results are inserted in a scale from positive (red) to negative (white) based on amplification time (t_amp; min:s). Real-time qPCR results are reported as positive (+) ore negative (−)

Fig. 3

Statistical correlation between LAMP amplification time (t_amp) and qPCR threshold cycle (C_t) obtained by testing with both methods each 1:5 serial dilution (ranged from 10 ng μL⁻¹ to 0.128 pg μL⁻¹) of each standard DNA template (X. fastidiosa—Co.Di.Ro strain; C. platani—isolate Cp24; P. ramorum-isolate Pram)