Detecting drug-resistant HBV and HCV variants using a DDS probe array Confidential PI: Shafer, David A., PhD PROJECT SUMMARY Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major global health concerns, with approximately one-third of the world’s population having been infected with either virus at one point in their lives. HBV and HCV infections lead to clinically indistinguishable symptoms, including liver cancer and cirrhosis. Serological or molecular diagnostics is necessary to identify the underlying etiological agent in either case. HBV can be cured by interferon therapy in some cases, although the success rate is low and adverse effects are common. HCV is most commonly treated with combination therapy with pegylated interferon and ribavirin. Recently, the WHO recommended the use of targeted antiviral compounds to treat chronic HBV and HCV infection. While they are safe and effective, drug-resistant stains have emerged. Thus, drug-susceptibility profiling is critical when selecting appropriate therapies against HBV or HCV. Point-of-care (POC) testing is important for making informed treatment decisions in a timely manner, increasing patient follow up, and (ultimately) reducing the spread of infection. However, current molecular diagnostics assays for drug-resistant HBV and HCV require expensive instrumentation and tests, making them relatively uncommon at the POC. The long-term goal of this Phase I SBIR application is to develop a simple probe array for endpoint analysis of PCR amplicons spanning common HBV and HCV resistance sites. The assays used to accomplish this goal will involve two of our real-time PCR probe technologies. Our internal DNA Detection Switch (iDDS) probes comprise a fluor-labeled probe component that matches a target-specific sequence, and a quencher- labeled antiprobe component that is nearly complementary to the probe. In the absence of the intended target, the probes and antiprobes bind together, thereby quenching probe fluorescence and preventing off-target detection. This system provides single-base discrimination over a wide temperature range (10–30ºC). Universal probes employ a generic probe and antiprobe that link to an extended primer, enabling low-cost multi-target detection. The Specific Aims of this proposal are (Aim 1), to develop Universal/iDDS probe assays for WT sequences at resistance sites in HBV and HCV, and (Aim 2) to develop a probe array for endpoint detection of HBV/HCV drug-resistance variants. Development of the probe array will facilitate simultaneous screening for drug-resistant HBV/HCV drug-resistance variants at 42 different codons in the HBV and HCV genomes. Successful completion of the Phase I proposal will justify subsequent Phase-II validation studies with clinical isolates to develop a final product containing lyophilized iDDS probes in 96-well format. In Phase II, we will also determine the assay’s linearity, analytical sensitivity, precision, analytical specificity...