A Bio-Nano Approach to Point-of-Care Testing of Parasitic DNA in Resource Limited Settings

Effective treatment and prevention of infectious diseases requires accurate diagnosis. Current diagnostics are expensive, time consuming, require sophisticated equipment and adequate infrastructure – requirements that are often not met in resource limited settings. There is a great need for simple and cost effective point-of-care tests to probe for infectious diseases.

We aim to develop a point-of-care diagnostic test that probes for the presence of pathogenic DNA in body fluids. The key scientific innovation is to use the CRISPR/Cas9 genome-engineering system as a DNA detection tool. The Cas9 protein and its sensing guide RNA have the unique capability to find a specific target DNA sequence. Initial proof-of-principle experiments show that our CRISPR/Cas9 model is able to successfully detect the target DNA sequence. For readout, we have designed a Cas9-guide complex that will be coupled to an enzymatic DNA molecule (DNAzyme) that will facilitate a colorimetric readout in the presence of the target DNA sequence.

To increase sensitivity in detection, we are further developing an approach that simultaneously detects the target DNA sequence and amplifies the readout. A “trigger” DNA sequence, displaced in the presence of the target DNA, will initiate a cleavage-based amplification cycle that will result in the release of multiple DNAzyme molecules. The hybridization between the trigger and seed sequence in the absence of the target DNA ensures to avoid false-positive results.

Upon completion, the developed device is expected to be adaptable to detect any pathogenic DNA sequence, simply by equipping the guide RNA with a new target. The device will not only benefit those living in resource limited settings, but also provide a cost effective alternative to current diagnostics in developed countries.