Simple, direct amplification of RNA-containing paper discs for diagnosing the hepatitis C virus

Abstract

Nucleic acid extraction (NAE) is crucial for molecular diagnostics but presents challenges in point-of-care testing (POCT) and decentralized settings. We developed a streamlined, paper-based NAE method for hepatitis C virus (HCV) RNA amplification, suitable for integration into POCT and lab-on-a-chip systems. This method uses Fusion 5 paper discs, completing extraction in under 30 min without centrifugation. The nucleic acids on the disc can be directly used or eluted for amplification. We validated this method's compatibility with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RQ-PCR), and loop-mediated isothermal amplification (LAMP), demonstrating versatility across amplification platforms. Clinical evaluation (n = 60) showed 100% sensitivity and specificity with a low detection limit of ~10¹ IU/mL. Results matched those from standard HCV RQ-PCR, confirming accuracy. Additionally, incorporating polyethylene glycol (PEG) improves extraction efficiency, eliminating the need for ethanol treatment and washing/drying steps. This modification enhances performance and suitability for field applications. Our paper-based HCV amplification is affordable and user-friendly, making it valuable for decentralized HCV detection and supporting global health initiatives.