Diagnostic power of one-step and two-step RT-qPCR methods to SARS‑CoV‑2 detection

Coronavirus-2019 (COVID-2019) is a novel coronavirus known as Acute Respiratory Syndrome (SARS-CoV-2). The premier standard test for SARS-CoV-2 diagnosis is a one-step RT-qPCR method, which requires specific probes and reagents. Therefore, detection on a large scale is expensive and cannot be very accurate.

A cost-effective technique based on SYBR green was evaluated in the current study. The specific primers for S and N genes were designed, then performed the cross-reactivity test with other coronavirus and respiratory viruses positive samples. Moreover, the analytical sensitivity test was carried out with 8 dilutions (1:10). Lastly, the SARS-CoV-2 clinical samples (n = 210) were tested by these two methods, and receiver operating characteristic (ROC) analysis was performed to investigate the incremental diagnostic value of each gene in the study methods.

The two-step method detected up to 6th dilutions of the SARS-CoV-2 samples and did not show any amplification of the positive samples of other respiratory viruses. ROC analysis revealed a diagnostic ability of the two-step method for SARS-CoV-2 with an area under the ROC curve of ≥ 0.7 (P ˂ 0.05) and relatively high sensitivity and specificity. The combination of N and S genes increased the sensitivity up to 88%, specificity up to 86%, and area under the ROC curve up to 0.85 (95% confidence interval (95% CI) 0.72 to 0.93, P = 0.0461).

Our findings indicated that the two-step method has comparable sensitivity and specificity to the one-step method. Therefore, this method can be considered a potential diagnostic method for diagnosing and monitoring COVID-19 patients. It suggests that when the one-step RT-qPCR method is not available, the two-step RT-qPCR can be used to identify SARS-CoV-2.