Sensitive, Single-Pot, Two-Stage Assay for Hepatitis Viruses

When left untreated, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections may cause severe illnesses. Since these infections remain asymptomatic for many years, routine screening of populations at risk is critical for therapy initiation. The current standard of care mandates a screening antibody test for HCV, followed by a confirmatory laboratory-based molecular test and treatment. Multiple visits to the clinic are inconvenient, and many patients fail to follow up. To address this challenge, we have developed sensitive, two-stage, isothermal molecular (Penn-RAMP) point-of-care tests to enable test and treat strategy. Penn-RAMP’s first stage is comprised of recombinase polymerase amplification (RPA), while its second stage is comprised of loop-mediated isothermal amplification (LAMP). Penn-RAMP is more sensitive than LAMP or RPA alone. We designed a custom pre-LAMP buffer to maximize the volume of RPA products that can be added to the LAMP reaction mix without inhibition and forward and backward primers. Penn-RAMP was implemented in a single pot comprised of two compartments separated by a thermally removable barrier. RAMP’s first stage is carried out above the barrier at the RPA incubation temperature. When the pot is heated to the LAMP incubation temperature, the barrier melts away, and the RPA reaction volume mixes with the pre-LAMP buffer, facilitating second-stage amplification. This entire process can be carried out with minimal instrumentation. Our HBV and HCV tests detect, respectively, as few as 10 and 25 virions within 30 min. The viral load can be estimated based on signal threshold time.