Simple and economical RNA extraction and storage packets for viral detection from serum or plasma

Abstract

AbstractRNA extraction is an essential step for detection and surveillance of common viral pathogens. Currently, sample processing and RNA extraction are costly and rely on proprietary materials that are difficult to acquire, maintain, and safely discard in low-resource settings. We developed an economical RNA extraction and storage protocol that eliminates the use of instrumentation, expensive materials, and cold chain requirements. Through an iterative process, we optimized viral lysis and RNA binding to and elution from glass fiber membranes. Protocol changes were evaluated by testing eluates in virus-specific real-time RT-PCRs (rRT-PCRs). Efficient, non-toxic viral lysis was achieved with a sucrose buffer including KCl, proteinase K and carrier RNA. Glass fiber membranes demonstrated concentration-dependent RNA binding of three arthropod-borne RNA viruses (arboviruses): dengue, chikungunya and Oropouche. Membrane binding was significantly increased in an acidic arginine binding buffer. For the clinical evaluation, 36 dengue virus (DENV)-positive serum samples were extracted in duplicate in the optimized protocol and results were compared to a commercial method. DENV RNA was successfully extracted from 71/72 replicates (98.6%) in the extraction packets, and rRT-PCR Ct values correlated between the techniques. Five clinical samples were selected to evaluate ambient-temperature storage up to 7 days on dried glass fiber membranes. DENV RNA was stable at 1, 3 and 7 days post extraction, with a mean difference in eluate RNA concentration of 0.14 log10 copies/μL. At a cost of $0.08 /sample, RNA extraction and storage packets address key limitations to available protocols and may increase capacity for molecular detection of RNA viruses.