MP102-LABINABAG: development of a prototype molecular diagnostic kit for the on-site detection of Macrophomina phaseolina using a portable PCR platform

The early diagnosis of plant pathogens from plant and soil samples needs a simple, rapid and cost-effective test, without the use of sophisticated and expensive equipment and reagents. In this study, the soil-borne pathogen Macrophomina phaseolina is used to demonstrate the potential for on-site molecular detection of the prototype diagnostic kit developed. This was achieved using a rapid and simple protocol comprising of a magnetic bead-based nucleic acid extraction, a portable PCR platform and a final NALFIA assay with a generic lateral flow device (LFD). The portable PCR platform powered by an external battery pack and all materials and reagents for an accurate on-site detection of M. phaseolina within 2 h were included in a professional bag. The DNA extraction method from infected plant and soil material can be completed within 45 min, and does not require centrifugation steps, organic solvents, or the use of liquid nitrogen for sample homogenization. A procedure for lyophilizing PCR reagents was developed to allow their storage and transportation at room temperature and their use in the field. Formulation development, trial freeze drying cycle and post-process analyses were performed in collaboration with the Biopharma Technology Company. DNA extracts were tested by PCR, which is completed in just 1 h, using patented primers for M. phaseolina labelled with biotin and FITC at the 5′ end. The results obtained were comparable to those of PCR testing in the laboratory. Research to develop this platform for the detection of other important plant pathogens is ongoing in our laboratory. This work was supported by the University of Pisa, Bando Dimostratori Tecnologici 2018, D.R.1937/18 del 6.11.2018, Progetto MP102- LABINABAG