An Isothermal Method to Amplify RNA from Bloodborne Viruses

PROJECT SUMMARY Bloodborne RNA viruses, in particular HIV-1 and hepatitis C, are a major public health burden. The diagnostic gold standard for these viruses is the use of RT-qPCR, which is expensive for use in developing countries. Traditional tests for HIV-1 also cannot be used to assess antiretroviral efficacy, as RT-qPCR could pick up integrated DNA and serological tests can detect antigens chromosomally expressed from integrated DNA. We propose the development of a diagnostic assay that will amplify and detect the RNA of bloodborne RNA viruses via a unique RNA amplification process. By developing a method to enrich viral RNAs over eukaryotic or bacterial RNAs we can lower the limit of detection and potentially reduce the level of false positives and negatives observed when using RT-based diagnostics. As this method is specific for viral RNA in general, it can be used for other human ssRNA viral pathogens, such as Zika or Dengue. The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project will be the development of a new step in viral RNA detection, usable for detecting the five bloodborne viruses we propose here, but also other ssRNA viruses. By using an isothermal approach to amplify viral RNA our device will eliminate the need for expensive equipment, which will increase access to testing. In the wake of the SARS-CoV-2 pandemic, the general public learned that Point-of-Care (POC) tests for antibodies or antigens do not detect low levels of viral infections. We propose to develop a test that will selectively amplify viral RNAs, which will bring the sensitivity of nucleic acid amplification to POC providers. The proposed work will validate the specificity of the enzyme toward viral RNAs, develop a system to extract and detect these RNAs, and lower the detection limit for POC diagnostics.