Clinical validation of innovative, low cost, kit-free, RNA processing protocol for RT-PCR based COVID-19 testing.

The current gold-standard molecular diagnosis for COVID-19 is based on a multi-step assay involving RNA-extraction and RT-PCR analysis for the detection of SARS-CoV-2. RNA-extraction step has been a major rate-limiting step in implementing high-throughput screening for COVID-19 during this pandemic. Moreover, clinical laboratories are facing several challenges that include cost, reagents, instrumentation, turn-around time, trained personnel, and supply-chain constraints to efficiently implement and sustain testing. Cognizant of these limitations, we evaluated the extraction-free methods described in the literature and have developed an innovative, simplified and easy protocol employing limited reagents to extract RNA for subsequent RT-PCR analysis. Nasopharyngeal-swab samples were subjected to the following individual conditions: 65{degrees}C for 15 minutes; 80{degrees}C for 5 minutes; 90{degrees}C for 5 minutes or 80{degrees}C for 1 minute, and processed for direct RT-PCR. These groups were also compared with a supplemental protocol adding isopropanol-ethanol-water elution steps followed by RT-PCR assay. The direct RT-PCR assay did not detect SARS-CoV-2 within the various temperature incubation only groups, whereas, the 90{degrees}C for 5 minutes-isopropanol-ethanol-water method was found to be comparable to the FDA-EUA method. Evaluation of the performance metrics for 100 clinical samples demonstrated a sensitivity of 94.2% and a specificity of 100%. The limit of detection was ascertained to be [~]40 copies/ml by absolute-quantification. The protocol presented for this assay employs limited reagents and yields results with high sensitivity. Additionally, it presents a simplified methodology that would be easier to implement in laboratories in limited resource countries in order to meet the high current COVID-19 testing needs.