PLANT-Dx: A Molecular Diagnostic for Point of Use Detection of Plant Pathogens

M. Verosloff; J. Chappell; K. L. Perry; J. R. Thompson; J. B. Lucks

PLANT-Dx works by first using recombinase polymerase amplification (RPA) (13) (Supplementary Figure 1) to amplify a target region of a plant pathogen genome to produce a double-stranded DNA construct that encodes the synthesis of a synthetic RNA regulator called a Small Transcription Activating RNA (STAR) (SI Figure 1 ) (11) . These DNA templates are then used to direct the transcription of STARs within a cell-free gene expression reaction (12) , which when produced, activates the transcription of a STAR-regulated construct encoding the enzyme catechol 2,3-dioxygenase (CDO) (14) (Figure 1A) . Only when the pathogen is present is the RPA product made, leading to expression of CDO, which in turn converts the colorless catechol compound into a visible yellow product. Here we show that this design can detect CMV in infected plant lysate with a low picomolar sensitivity, and can be configured to detect nucleic acids from different viral genomes without crosstalk. In addition, we show that this design requires only simple mixing and body heat to induce a color change, which we anticipate will facilitate deployment to field settings.