Towards the development of point-of-care electro-catalytic biosensors for diagnostics of Botrytis spp. of the temperate legumes

Bilkiss M1, Bar I1, Brownlie J1, Shiddiky MJA1,2 and Ford R1

  1. School of Environment and Science, Griffith University, Nathan Campus, QLD 4111, Australia.
  2. Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan Campus, QLD 4111, Australia.

Botrytis grey mould (BGM), caused by Botrytis cinerea and Botrytis fabae, separately or within a complex, causes serious yield losses in legumes during conducive seasons in Australia and worldwide. A greater success in Integrated Disease Management (IDM) approaches to prevent this loss would result from fast, accurate and cost-effective diagnosis and quantification of the causal pathogen(s). Tools to enable this would provide a large opportunity to save on targeted fungicide chemistries input costs and enable the faster application of other management options. The existing immunogenic and molecular probe type diagnostic methods, based on whole genome sequencing, PCR amplification or antibodies, are time consuming and offer varying levels of specificity and/or sensitivity. As an alternative, we have developed species-specific molecular biosensors for the fast, accurate, sensitive detection and quantification of the mycelium and spore derived nucleic acid of both of the target pathogens. For this, two sets of species-specific primers, Bc-F/Bc-R for Botrytis cinerea and Bfa-F/Bfa-R for Botrytis fabae, were designed. Initially, probe sensitivities were determined (100 fg/μl equivalent to 2 genome copies/μl) using multiplexed quantitative PCR. Simultaneously a specific and sensitive assay for the electro catalytic detection of the target pure fungal DNA using functionalised magnetic nanoparticles has been accessed and showed 100 times more sensitivity than the quantitative PCR towards the development of point-of-care diagnostic biosensors for Botrytis spp. of the temperate legumes.