Development and validation of a new triplex real-time quantitative reverse Transcriptase-PCR assay for the clinical detection of SARS-CoV-2

Abstract

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.

Keywords: LDT; ORF8; RNaseP; SARS-CoV-2; Spike; Variants of concern.

Fig. 1

Distribution of C_T's recorded between the RNaseP and mRNaseP TaqMan™ assays for a cohort (n = 40) of SARS-CoV-2 negative and positive NPFS samples. ΔC_T = C_T (mRNaseP)-C_T (RNaseP). A two-sided paired-sample t-test found statistically significant difference between (***P < 0.001) the CT values obtained between RNaseP and mRNaseP TaqMan™ assays, as shown by P values. Dotted line indicates the assay cut-off at C_T ≤ 40 as defined in materials and methods.

Fig. 2

Correlation between C_T values obtained between (A.) SORP triplex assay and BCCDC triplex assay (R²:0.99; Spearman's ρ = 0.96; P < 0.0001) (B.) SORP triplex assay and N1/N2 US-CDC Nucleocapsid singleplex assay. (R²:0.99; Spearman's ρ = 0.97; P < 0.0001).