The implementation of a rapid sample preparation method for the detection of SARS-CoV-2 in a diagnostic laboratory in South Africa

Abstract

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

Fig 1. Comparison of target Ct values after RSP and NucliSENS® easyMag NA purification.

The Ct values for the SARS-CoV-2 (A) Envelope (E), (B) RNA-dependent RNA-polymerase (RdRp) and (C) Nucleocapsid (N) gene targets are shown for samples tested with the Allplex™ 2019-nCoV assay after NucliSENS® easyMag® NA purification and RSP. The difference in generated Ct values was found to be statistically significant in each case with a P value of <0.0001 as determined by paired t-test.

Fig 2. Comparison of target Ct and CN values after RSP and testing with the Abbott RealTime SARS-CoV-2 assay.

The Ct values for the SARS-CoV-2 Envelope (E), RNA-dependent RNA-polymerase (RdRp) and Nucleocapsid (N) gene targets are shown for samples tested with the Allplex™ 2019-nCoV assay after RSP and CN values after testing with the Abbott RealTime SARS-CoV-2 assay. A plotted CN or Ct value of 40 indicates that detectable amplification did not occur. The Abbott assay CN values are assay specific and not directly comparable to Ct values, but are shown to demonstrate the performance of the spectrum of selected samples.