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Multiplex detection of plant pathogens using a microsphere immunoassay technology.

Ratthaphol Charlermroj1, Orawan Himananto, Channarong Seepiban

  • 1Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom. rcharlermroj01@qub.ac.uk

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A new microsphere immunoassay can rapidly and accurately detect four major plant pathogens simultaneously. This method offers higher sensitivity and a shorter assay time than traditional ELISA, improving plant disease control.

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Area of Science:

  • Plant Pathology
  • Immunology
  • Biotechnology

Background:

  • Plant pathogens pose significant threats to global agriculture, impacting seed export, disease management, and quarantine efforts.
  • Rapid and accurate detection methods are crucial for preventing the worldwide spread of plant diseases.
  • Current detection methods can be time-consuming and lack the sensitivity required for effective screening.

Purpose of the Study:

  • To develop a novel multiplex detection method for simultaneously identifying four key plant pathogens.
  • To enhance the sensitivity and speed of plant pathogen screening compared to existing techniques.
  • To validate the efficacy of the new method in both laboratory and natural plant infection scenarios.

Main Methods:

  • Development of a multiplex microsphere immunoassay utilizing fluorescence-coded magnetic microspheres.
  • Immobilization of specific antibodies onto microspheres for capturing target pathogens: Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV).
  • Detection of captured pathogens using R-phycoerythrin (RPE)-labeled antibodies, with optimization of assay conditions including antibody pairs, blocking buffers, RPE concentration, and incubation times.

Main Results:

  • The optimized microsphere immunoassay accurately detected all four target plant pathogens simultaneously.
  • The assay demonstrated significantly higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when tested with spiked samples and healthy watermelon leaf extracts.
  • The total assay time was reduced to 1 hour, compared to 4 hours for ELISA.
  • The method successfully detected pathogens in naturally infected plant samples.

Conclusions:

  • The developed microsphere immunoassay is a highly sensitive, rapid, and accurate method for multiplex plant pathogen detection.
  • This novel system represents a significant advancement in plant disease diagnostics, aiding in seed export, disease control, and quarantine.
  • The method's efficiency and speed offer substantial benefits for global plant health management and biosecurity.