Visualizing in-field detection of HCV using a one-pot RT-RAA-CRISPR/Cas12a platform

Hepatitis C, one of the major infectious diseases posing a serious threat to human health, contributes a significant disease burden to global public health governance. Low diagnostic rates are a major barrier to eliminating hepatitis C in resource-constrained countries. As a result, the development of rapid, accurate, ultra-sensitive, and user-friendly POCT assays is desperately needed to improve the diagnostic rate and control of HCV. Here, we present a Visual One-Pot RT-RAA-Cas12a (HCV-VOpRCas12a) platform, which performed RT-RAA and CRISPR-based detection in a single tube by physical separation, and low-cost, readily accessible hand warmers were used as incubators. A visualization device was built to achieve the visual readout. The LoD of the HCV-VOpRCas12a platform was as low as 10⁰ copies per μL and only took about 15 min to achieve HCV-RNA diagnosis. In the validation of 101 clinical serum samples, the detection sensitivity and specificity of the visualization device were 95% and 100%. The VOpRCas12a platform holds enormous potential in achieving a global strategy to eliminate the public threat of HCV infection by 2030 and in the next generation of real-time molecular diagnostics.