Details
Project title: Development of an innovative plant diagnostic system based on separation by photoactive magnetic nanoparticles and fluorescence detection
Acronym: FLUOMAG
Coordinator: Giuseppe Caputo (Pianeta s.r.l.)
TP/LS membership: CMDI/NDHCA1
Subjects involved: Cori, National Institute of Metrological Research, Institute of Plant Virology, University of Turin (NIS)
Status: Completed
Abstract: The FLUOMAG project enabled the design, study and prototyping of a diagnostic system
innovative molecular based on the use of magnetic nanoparticles as solid phase separation, new compounds
fluorescent for the detection of signal and oligonucleotidal probes specially studied and generated by techniques of cloning representative plant diseases such as the flavescence dorée of the vine and the chrysanthemum yellowness. The diagnostic system has proved industrializable and, in the future, marketable.
Contact for further information:
Name: Giuseppe Caputo
Organization: Pianeta S.r.l.
Address: Via Giannone 3, 10036 Settimo Torinese (TO)
Phone: 011.8028437
E-mail: giuseppe.caputo@pianeta.eu
Web: www.pianeta.eu
THE PROBLEM ADDRESSED
Currently one of the main problems of plant diagnostics is the availability of methodologies that allow
rapid and repeatable diagnosis of microbial population interacting with the plant, as well as characterization in only one
testing of the set of such microorganisms. This is all the more important when we consider that, for many species of
agronomic interest, several pathogenic and/or symbiont micro-organisms are not isolatable by classical cultivation methods.
Superparamagnetic particles, with the possibility of activation of their surface with different molecules (nucleic acid probes or antibodies) and with the design of specific fluorescent compounds represent a possible solution to the problems described. They are already successfully used in the medical field where specific protocols are already available for the introduction of reactive groups on the surface that allow the subsequent covalent bond with active molecules, especially antibodies.
Rapid bio-barcode tests for the specific detection of synthetic DNA in multiplex format have also recently been developed. These procedures are adaptable to the detection of the genomic DNA of micro-organisms (pathogens and not) and, in the case of micro-organisms present in very poor concentration, it can be hypothesized to use the messenger RNA of highly expressed genes as a diagnostic target.
Moreover, the production of these particles can be carried out within one of the Institutes involved in this project and does not present particular difficulties. Currently the kits on the market have a low reliability and a high cost, as well as characteristics not specific for the phytosanitary use.
THE ACTIVITIES CARRIED OUT
The research project was divided into five technical workpackages (WP) grouping homogeneous activities.
WP1 “Identification and design of diagnostic reagents” addressed the problems in the identification of diagnostic targets (virus and phytoplasms of the vine)the definition of the parameters of synthesis of the materials used and the specifications of the diagnostic system as a whole.
In WP2 “Development and implementation of magnetic nanoparticles” the production of magnetic nanoparticles (NP) functionalized by the following steps was performed:
- synthesis of superparamagnetic iron oxide nanoparticles;
- coating of particles produced with an amorphous silica layer;
- surface functionalization with amino groups;
- activation with a hetero – bifunctional reagent (sulfo-SMCC);
- conjugation of the functionalized particles with probe oligonucleotides capable of recognizing sequences of the genetic material of the pathogen of interest.
In the WP3 “Design and synthesis of reagents” the sequences of oligonucleotides that are used in the diagnostic test were designed and obtained. The obtained oligos were then suitably functionalized to allow the binding to the magnetic NP (capture probes) and fluorescent compounds that have been designed and synthesized for the detection of the hybridization signal between the capture probe and the target sequence.
In the WP4 “Optimization of diagnostic protocols” many components preparations were made in small volumes, used immediately after preparation to carry out laboratory tests (operation tests)essential to achieve the best possible formulation of the kit.
Finally, in WP5 “Prototyping and testing”, we started from the definition of packaging parameters: materials, volumes, etc. keeping in mind the needs of future users of the kits: number of tests per package, modularity of the product (that is how many different sessions of analysis can be carried out with the volume of reagents provided), convenience and safety of use, etc. The panel of samples of CV Barbera vines infected with flavescence dorée from a vineyard in production has been identified and diagnostic checks for phytoplasms have also been completed. With the support of the technical documentation, the prototype kits were prepared, necessary for the final validation and stability tests
RESULTS ACHIEVED AND EXPLOITATION OF THE RESULTS
The objectives of the Project have been achieved. The templates for the diagnosis, the design of the probes and the functionalization of the magnetic nano particles have been successfully carried out demonstrating that the method followed is sufficiently flexible to be adapted to the diagnosis of different nucleic acid molecules, paving the way for further developments in the areas of diagnosis and detection of analytes consisting of chains of different lengths of ribonucleic and deoxyribonucleic acid.
The three devices that the prototypes have been prepared for and validated have shown high performances that make us think positively about the possibility of their success on the market. The data obtained with the two devices of which the development has been realized, but not the preparation of the prototypes, authorize optimism about the performances that can provide and that will be verified, with activities external to the project, when the necessary resources are available. The data collected so far in relation to the model made with the methodology based on the immobilization on magnetic nanoparticles of nucleotide probes specific to the pathogens considered, through a molecular recognition process, Magnetic separation and detection with fluorescent oligonucleotidic markers, demonstrate the feasibility of the device.
The Project has also strengthened the link between the different Partners who have actively collaborated to achieve the results.
PROJECT NUMBERS
- Other Private Partners: : Cori s.r.l.
- Other Public Partners: INRIM, CNR-IVV, Unito-NIS
- Total number of partners: 5
- Duration in months: 36
- Total budget: 546.000 euro
- Funding: 273.000 euro
- Number of scientific publications: 2
- Number of presentations at conferences and seminars: 4
- Number of patents filed: 1
- Number of permanent, fixed-term and cocopro jobs created: 3
- Number of jobs retained at the end of the project: 1
- Number of public researchers involved: 12
