A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR

Abstract

The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step RT-qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step RT-qPCR against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by RT-qPCR with high sensitivity (~4 viral RNA copies per RT-qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.

Keywords: RNA extraction; RT-qPCR; SARS-CoV-2; SARS-CoV-2 RT-qPCR; clinical microbiology; molecular microbiology; rapid diagnostic methods; virology; virus inactivation.

Figure 1

The effect of GITC salts on the efficiency of a two‐step B2M RT‐qPCR using dilutions of the aqueous phase from the GPC extraction of an NP and OP sample. A representative amplification plot of ΔRn against PCR cycle number for the two‐step B2M RT‐qPCR with different dilutions of the RNA‐GITC solution in the RT reaction (ranging from 8× to 100×). No amplification was observed at 8× and 20× dilutions. The threshold is shown as a black dashed line and corresponds to 0.116 for the NP swab and 0.040 for the OP swab. ΔRn: Rn (the fluorescence of the reporter dye divided by the fluorescence of the passive reference dye ROX) minus the baseline (black dashed line). The amplifications were performed in duplicate.

Figure 2

The effect of GITC and other salts on the efficiency of SARS‐CoV‐2 RT‐qPCR and assay sensitivity. (A) The effect of GITC and other salts on SARS‐CoV‐2 RT‐qPCR detection of virus in the diluted aqueous phase of samples from a COVID‐19 patient. Cycle threshold (Ct) for RT‐qPCRs targeting the N gene on the SARS‐CoV‐2 virus RNA, with viral RNA extracted from an OP swab from confirmed COVID‐19 patient. The internal control RNase P RT‐qPCR amplifications were negative at 50× and 75× dilutions (not shown). (B) Determination of the sensitivity of the SARS‐CoV‐2 one‐step RT‐qPCR protocol using synthetic SARS‐CoV‐2 RNA combined with RNA extracted from an OP swab from a healthy individual using the GPC‐extraction method. The concentration of GITC and other salts were constantly kept at a 100× dilution in each of the diluted virus samples. Cycle threshold (Ct) for RT‐qPCRs targeting the N gene on the synthetic SARS‐CoV‐2 virus RNA using the N1 and N2 primer sets, respectively. Amplification of RNAse P was used as an internal control. The amplifications were performed in duplicate.

Figure 3

Workflow for SARS‐CoV‐2 detection using the simplified GPC‐extraction method.