An Open One-Step RT-qPCR for SARS-CoV-2 detection

Abstract

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, and the kit showed comparable sensitivity to approved commercial kits. The One-Step RT-qPCR was then tested on clinical samples and demonstrated similar performance to commercial kits in terms of positive and negative calls. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

Keywords: Diagnostic system; One-Step RT-PCR; Open Source; Recombinant enzymes; SARS-CoV-2.

Figure 1:. CDC SARS-CoV-2 N1 and N2 probe-based One-Step RT-qPCR assays performed with synthetic RNA using homebrew M-MLV RT and Taq DNA pol.

(A-B) Representative amplification curves using N1 and N2 CDC-approved double-quenched probes. Each curve represents a specific dilution of SARS-CoV-2 synthetic N RNA used as template: 1.4 × 10⁷ copies approximately (red line), 1.4 × 10⁶ (yellow line), 1.4 × 10⁵ (green line), 1.4 ×10⁴ (light blue line), 1.4 × 10³ (blue line), 1.4 × 10² (purple line), 1.4 × 10¹ (light brown line) and no template control (NTC, gray line). Amplification curves for synthetic N RNA from MERS-CoV (bla ck dashed line) and SARS-CoV-1 (orange dashed line) are also indicated. Characteristic Cq values are indicated on the upper left side of each panel. N.d.: non-detected (no Cq reported).

Figure 2:. Probe-based Open One-Step RT-qPCR reaction mix (M-MLV RT and Taq DNA pol) provides comparable results to commercial kits in SARS-CoV-2 clinical samples.

Scatterplot of the Cq values of positive (blue circles) and negative (red squares) samples obtained by the commercial and open RT-qPCR reaction master mixes using the N1 and N2 primer/probe sets (A and B, respectively). The numbers displayed in each sample match those displayed in Table 3 (# Sample). If a Cq value was not detected in the sample, it appears in the ND area of the graph depending on whether this occurred in the commercial kit (green rectangle), the open probe-based kit (gray rectangle), or both (intersections between the rectangles). For each combination of primers and probes, the linear trend of the positive samples is shown (blue dotted line) along with the corresponding value of r². ND: non-detected