Development of point-of-care and multiplex diagnostic methods for the detection of plant pathogens

Abstract

New diagnostic technologies for the detection of plant pathogens with high multiplexing ability and point-of-care (POC) capability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. The aim of this project is to develop new methods combining nucleic acid amplification, multiplexing and point-of-care application for the detection of plant pathogens. Isothermal amplification methods, such as recombinase polymerase amplification (RPA) which operates at low constant temperature are especially suited for POC applications. However, the development of a rapid and simplified detection method for the isothermal amplification products in resource-poor settings is still challenging. A new method to visualize the success of the amplification step, and therefore the presence of pathogen DNA in a sample, was developed based on bridging flocculation. This method requires minimal equipment and can be assessed by the naked eye allowing its use in POC settings. The method was initially applied to the rapid and sensitive detection of several important plant pathogens and subsequently extended to animal and human pathogens to demonstrate the wide applications of the approach. A nanoparticle based electrochemical biosensor was also developed for rapid and sensitive detection of amplified plant pathogen DNA on screen-printed carbon electrodes. Gold nanoparticles were employed as a probe for electrochemical assessment by differential pulse voltammetry (DPV). This method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. To allow multiplex detection of plant pathogen, a novel method that can screen for thousands of plant pathogens with high specificity and sensitivity using molecular inversion probes (MIPs) was been developed. As proof of concept, a MIP targeting a unique DNA sequence present in the F.oxysporum f.sp. conglutinans (Foc) genome was designed. The specificity, sensitivity and detection limit of the assay were assessed and used to detect the presence of pathogen in infected Arabidopsis thaliana samples. This methodology successfully detected as little as 2.5 ng of pathogen DNA and it was highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. Finally, a diagnostic platform for POC plant pathogen detection using a combination of surface-enhanced Raman scattering (SERS) and RPA was developed for rapid multiplex detection. The RPA-SERS method was faster, more sensitive than polymerase chain reaction and could detect as little as 2 copies of B. cinerea DNA. Furthermore, multiplex detection of Fusarium oxysporum f.sp. conglutinans, f.sp. lycopersici and Botrytis cinerea was demonstrated in vivo in Arabidopsis thaliana and tomato. The assay was subsequently simplified into a single tube assay and tested outside of a laboratory setting to detect pathogens in tomato plants.