Standard diagnoses of SARS-CoV-2 infections are done by RNA extraction and real-time RT-PCR (rRT-PCR). However, the need for RNA extraction complicates testing due to increased processing time, high cost, and limited availability of commercial kits. Therefore, alternative methods for rRT-PCR detection of SARS-CoV-2 without RNA extraction were investigated. Nasopharyngeal and sputum samples were used to compare the sensitivity of three techniques: Trizol RNA extraction, thermal shock, and the direct use of samples with an RNase inhibitor. Direct, extraction-free use of primary samples plus the RNase inhibitor produced diagnostic values of 100% sensitivity and specificity compared to standard protocols, and these findings were validated in a second, independent laboratory.

RNA 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/uL. 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. Keywords: ribonucleic acid, extraction, dengue virus, molecular testing

RNA extraction methods and ultra-cold storage requirements limit molecular testing for common viruses. We developed a simple, flexible, and economical method that simultaneously addresses these limitations.

Since the beginning of the SARS-CoV-2 pandemic, supply chain shortages have caused major disruptions in sourcing the materials needed for laboratory-based molecular assays. With increasing demand for molecular testing, these disruptions have limited testing capacity and hindered efforts to mitigate spread of the virus and new variants. Here we evaluate an economical and reliable protocol for the extraction and short-term ambient temperature storage of SARS-CoV-2 RNA. Additional objectives of the study were to evaluate RNA from this protocol for 1) detection of single nucleotide polymorphisms (SNPs) in the spike gene and 2) whole genome sequencing of SARS-CoV-2. The RNAES protocol was evaluated with residual nasopharyngeal (NP) samples collected from Emory Healthcare and Emory Student Health services. All RNAES extractions were performed in duplicate and once with a commercial extraction robot for comparison. Following extraction, eluates were immediately tested by rRT-PCR. SARS-CoV-2 RNA was successfully detected in 56/60 (93.3%) RNAES replicates, and Ct values corresponded with comparator results. Upon testing in spike SNP assays, three genotypes were identified, and all variant calls were consistent with those previously obtained after commercial extraction. Additionally, the SARS-RNAES protocol yield eluate pure enough for downstream whole genome sequencing, and results were consistent with SARS-CoV-2 whole genome sequencing of eluates matched for Ct value. With reproducible results across a range of virus concentrations, the SARS-RNAES protocol could help increase SARS-CoV-2 diagnostic testing and monitoring for emerging variants in resource-constrained communities.