A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2

Abstract

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

Keywords: COVID-19; RNA; RT-qPCR; RdRP; SARS-CoV-2; SYBR Green; TaqMan; diagnosis.

Figure 1.

Selection of a primer pair for SARS-CoV-2 detection. (A) Serial dilutions of a pool of positive samples for each primer pair. Representative amplification (left panel) and melting curves (middle panel) are shown. Corresponding RT-qPCR products were solved by gel electrophoresis and representative images of positive samples (+) and negative controls (-) are shown (right panel). (B) Summary of the data generated for selection of a highly specific primer pair. Ct: cycle threshold. Amplification efficiency and R² relates to the log-linear fit of the relation between Relative quantity vs. Ct for known serial dilutions of a positive control. An amplification efficiency of 100% represents a decrease of one Ct for a 1:2 dilution. Unspecific amplification: Ct of amplification curves observed in negative controls (without template), probably due to primer dimers. (C) UCSC Genome Browser image, depicting the position of the mutations within each SARS-CoV-2 variant of concern, and the position of the selected primer pair (RdRP) for the following experiments

Figure 2.

Using an intercalating dye can yield similar sensitivity and specificity as TaqMan probes. (A) Paired boxplot of the Ct shift for the SYBR Green RT-qPCR (RdRP amplicon) versus two different TaqMan-based reactions (N and ORF1ab amplicons) using the Charité protocol. (B) RT-qPCR amplification curves for serial dilutions of a SARS-CoV-2 standard RNA, from 4 × 10⁵ copies per µL (c/µL) to 4 c/µL, using either the unmodified Charité protocol (TaqMan) or the one adapted for an intercalating dye (SYBR Green). (C) Summary of the performance obtained using the different commercial and customized RT-qPCR protocols evaluated. (D) Ct values for each sample tested with every RT-qPCR protocol developed and optimized in this work. Not all samples were evaluated with all protocols. The cut-off for positivity for each mix is shown with an orange line. Diagnostic was done with a TaqMan commercial kit. Negative samples with undetermined Ct were assigned a pseudo-Ct of 41. (E) A drop in diagnostic sensitivity using two-step RT-qPCR approaches is circumscribed to samples with higher Ct values. Table summarizing the sensitivity observed using either the INBIO master mix or the custom-made mix when separating the positive samples in terciles (T1-T3) according to the previously assessed Ct value using the RT-qPCR DisCoVery kit (ORF1ab and N amplicons). (F) RT-qPCR amplification curves for either serial dilutions of a pool of positive samples (top panel) or random samples (bottom panel), using four different retro-transcriptases for cDNA synthesis

Figure 3.

Selection of a human housekeeping gene to be used as internal control. (A) Representative amplification curves for POLR2A and U1 using both the custom-made mix and the INBIO master mix. (B) Agarose gel electrophoresis of the products obtained after qPCR of 1:1, 1:16 dilutions and the negative control respectively, for each primer pair using both mixes