Detection of HIV-1 with RCA-Rcrispr on Smartphone

Close to 60% of people living with HIV remain undiagnosed in resource-limited areas. Making HIV a significant global health challenge, over 40 years after its discovery. HIV disproportionately affected the underserved and resource-limited community, despite all the efforts that have been made in the prevention and treatment of this disease. Thus, the development of better tools that are simple, accurate, and accessible, and do not rely on expensive equipment, is crucial to addressing this issue.

To bridge this gap, we have developed a low-cost, label-free RCA–rCRISPR diagnostic platform that can detect HIV viral load with minimal instrumentation. Our method combines an RNA-detecting rolling circle amplification (RCA) reaction with a plasmid reporter-based ratiometric CRISPR (rCRISPR) readout. This setup allows us to detect unprocessed RNA targets without the need for heavy pre-treatment steps. Using this approach, we were able to detect HIV RNA down to single-digit attomolar levels (~3000 copies/mL) from real patient samples ex vivo, providing a simple, accurate, and accessible solution.

Different from conventional RCA, we take advantage of the three activities of phi29 polymerase: exonuclease activity, polymerization, and strand displacement, which allows us to detect the entire genome directly.

For point-of-care use, we have gone a step further by developing a smartphone-based minigel electrophoresis device. Our device allows viral load testing in an accessible, equipment-free manner, which is especially crucial for communities with limited resources. Furthermore, our diagnostic platform is not limited to HIV detection. We have demonstrated its ability to detect point mutations, such as a BRAF mutation in canine urothelial carcinoma. This versatility underscores the potential of our approach for a wide range of diagnostic applications.