Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing

Abstract

Background: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2.

Objectives: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay.

Materials and methods: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes).

Results: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media.

Conclusions: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing.

Limitations: Small patient sample size.

Conflict of interest: None.

Figure 1.

Amplification curves produced by RT-qPCR for healthy donor samples prepared from nasopharyngeal swabs submerged in transport medium and spiked with MS2 and ECMV with step-down titrations. The y axis is ΔRn (the normalized reporter value, also called “Rn value”, of an experimental reaction minus the “Rn value” of the baseline signal generated by the instrument) and the x axis are reference cycles. rRNA is the human housekeeping gene, MS2 is a positive-sense single-stranded RNA bacteriophage and EMCV is the encephalomyocarditis virus, a small non-enveloped single-stranded RNA virus.

Figure 2.

Amplification curves produced by RT-qPCR for healthy donor samples prepared from nasopharyngeal swabs submerged in transport medium and spiked with heat inactivated lab-propagated SARS2-COV-2 viral particles. The S, N, and ORF1b gene amplification curves were produced after automated RNA extraction of decreasing titrations of SARS-COV-2 (from 15000 copies/test to ~60 copies/test followed by one-step RT-qPCR using the TaqPath assay by Applied Biosystems. The E, RdRp2, and RdRp4 gene amplification curves were produced after automated RNA extraction of decreasing titrations of SARS-COV-2 (from 3000 copies/test to 8 copies/test followed by one-step RT-qPCR using the in-house assay. The y axis is ΔRn and x axis are quantification cycles.

Figure 3.

Amplification curves produced by RT-qPCR for SARS-COV-2 samples obtained after diagnostic tests were performed (using leftover specimens), and prepared from nasopharyngeal swabs submerged in transport medium. The S, N, and ORF1b gene amplification curves were produced after automated RNA extraction SARS-COV-2 by one-step RT-qPCR using the TaqPath assay by Applied Biosystems. The samples were spiked by MS2, the internal control for RNA extraction. The y axis is ΔRn and x axis are quantification cycles.