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

Chiara Aglietti,Aida Raio,Paola Bartolini,Alberto Santini,Francesco Pecori,Alessia Lucia Pepori,Nicola Luchi,Paolo Capretti

doi:10.1186/s13568-019-0774-9

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.

Highlights

Invasive alien species represent a primary threat to biodiversity, economy and human health
Specificity of real‐time loop mediated isothermal amplification (LAMP) assay For each target pathogen (X. fastidiosa, C. platani and P. ramorum) the nucleotide–nucleotide BLAST ® search showed a complete homology (100%) between the LAMP amplicon sequences designed in the current study and the sequences of the same pathogen available in GenBank database (NCBI)
To our best knowledge no previous LAMP assay has been developed for C. platani. quantitative PCR (qPCR) showed higher sensitivity with respect to LAMP in X. fastidiosa and C. platani detection, while for P. ramorum limit of detection (LOD) was the same as that of LAMP

Summary

Introduction

Invasive alien species represent a primary threat to biodiversity, economy and human health. The high specificity and sensitivity of molecular DNA-based technologies allows detection of pathogens in the early stages of infection, when they are present at low DNA concentrations (Bilodeau et al 2007; Chandelier et al 2006; Harper et al 2010; Luchi et al 2013; Rollins et al 2016). Many of these methods have been used routinely in the laboratory, most of them are not transferable for field inspection, seriously limiting their adequacy for point-of-care application (Lau and Botella 2017). The use of portable detection instruments is a major driving force to achieve point-of-use, and real-time monitoring of analysed samples, allowing rapid detection

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Conclusion