A strategy to develop one step real-time RT-PCR for diagnosis of SARS-CoV-2 infection from clinical samples

Abstract

Abstract The aim of this study is to develop a one-step real-time PCR assay for SARS-CoV-2 detection. A lysis solution was prepared using Tween-20, Triton X-100, EDTA, and tris buffer (pH 7.4) and various parameters were optimised. Adding carrier molecules [Poly (A), glycogen, and linear polyacrylamide] to the lysis solution significantly improved RT-qPCR efficacy. Poly (A) was the most effective of all carriers. Diagnostic potential of this Poly (A) solution was demonstrated using 150 positives and 200 negative swabs, and the sensitivity of the RT-qPCR diagnostic test was estimated to be 98.6 (95%CI; 96.0, 101.17, p < 0.001) for group 1; Ct ≤ 25 and 87.2 (95%CI; 80.2, 94.0, p < 0.001) for group 2; Ct ≥ 25–30, with excellent accuracy (0.9 < AUC < 1.0), and 100% specificity.