Emergency SARS-CoV-2 variants of concern: rapidly direct RT-qPCR detection without RNA extraction, clinical comparison, cost-effective, and high-throughput

Abstract

Since the late 2020, the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has been characterized by the emergence of spike protein mutations, and these variants have become dominant worldwide. The gold standard SARS-CoV-2 diagnosis protocol requires two complex processes, namely, RNA extraction and real-time reverse transcriptase polymerase chain reaction (RT-PCR). There is a need for a faster, simpler, and more cost-effective detection strategy that can be utilized worldwide, especially in developing countries. We propose the novel use of direct RT-qPCR, which does not require RNA extraction or a preheating step. For the detection, retrospectively, we used 770 clinical nasopharyngeal swabs, including positive and negative samples. The samples were subjected to RT-qPCR in the N1 and E genes using two different thermocyclers. The limit of detection was 30 copies/reaction for N1 and 60 copies/reaction for E. Analytical sensitivity was assessed for the developed direct RT-qPCR; the sensitivity was 95.69%, negative predictive value was 99.9%, accuracy of 99.35%, and area under the curve was 0.978. This novel direct RT-qPCR diagnosis method without RNA extraction is a reliable and high-throughput alternative method that can significantly save cost, labor, and time during the coronavirus disease 2019 pandemic.

Keywords: COVID-19; SARS-CoV-2 VOC; cost-effective diagnosis; direct RT-qPCR; high-throughput.

Figure 1

Detection of SARS-CoV-2 using two methods on two different machines. Data are depicted as scattered dot plots with stated mean values. Each dot represents one C_t value corresponding with one specimen and p-values were found using paired t-test. (A) Shows SARS-CoV-2 N1 gene results. (B) Shows SARS-CoV-2 E gene results.

Figure 2

Correlation between cycle threshold values of N1 (A) and E (B) genes using conventional RT- qPCR and lab-developed RT-qPCR on LC96 or AIO. The correlation coefficient of the analysis on LC96 is R² = 0.9787 for N1 gene and 0.9524 for E gene. The correlation coefficient of the analysis on AIO is R² = 0.9850 of N1 and 0.9798 of E gene.