Portable hepatitis C virus RNA extraction and stabilization using low-cost lab-on-a-chip style components with shelf stable reagents

Abstract

Molecular tests offer a powerful approach to diagnosing numerous diseases and, due to advances in lab-on-a-chip technologies, have the potential to be used in the field and at the bedside. However, there is still a need for portable technologies that help isolate nucleic acids from their associated sample for testing. Previously, our groups developed an RNA extraction and stabilization (RNAES) protocol that utilized novel shelf stable reagents to extract, isolate, and store nucleic acids on membranes at ambient temperature. Building on these results, we have created RNAES kits with reductionist, 3D-printed, lab-on-a-chip style parts that enable field isolation of RNA without external equipment or power. Our kits feature three components, a lysis sphere, a capillary-wicking based cartridge for RNA isolation, as well as a drying & storage tube that stabilizes RNA for ambient temperature shipping. Using our kit, a user can process a 25 µL sample of serum or plasma within 15 minutes, including a 10-minute incubation time. Noting that hepatitis C virus (HCV) can be cured when detected, we use our RNAES kit in combination with a new real-time RT-PCR to detect HCV. Our data on 98 clinical samples and four contrived whole blood samples show comparable RNA detection and quantified viral loads following extraction with our RNAES kit or a commercial system. Our approach also provided the additional benefit of stabilizing viral RNA for storage at ambient temperatures as lysate, for up to 7 days, or on dried membranes in storage tubes, for at least 30 days.