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

We began by investigating the ability of PLANT-Dx to detect the presence of in vitro transcribed (IVT) RNA designed to mimic specific target regions of cucumber mosaic virus (CMV). We observed rapid color accumulation in samples containing 1 nM of purified transcription product versus the no-RNA negative control (Figure 1B) . To test for modularity, we further developed sensors and primer sets for the detection of potato virus Y (PVY), and confirmed function with the same assay ( Figure 1C) . The specificity of our system was also tested by interrogating the crosstalk between the product of various RPA reactions and noncognate molecular sensors. Specifically, we tested color production from cell-free reactions containing the reporter DNA construct for CMV with the PVY IVT-derived RPA product, as well as the converse, and found color production only between cognate pairs of input RPA and reporter constructs ( Figure 1D) . We next interrogated the inherent limit of detection of our system through titration of input IVT products ( Figure 1E ). This demonstrated our ability to detect the presence of target nucleic acid sequences down to the picomolar range. Surprisingly, this sensitivity is lower than that previously reported for RPA (3) and is most likely due to loss in amplification efficiency from the addition of the long overhangs present within our primer sets.