Diagnosis of SARS-CoV-2 during the Pandemic by Multiplex RT-rPCR hCoV Test: Future Perspectives

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a significant threat to public health. However, among the Coronaviridae family members, there are other viruses that can also cause infections in humans. Among these, severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV) have posed significant threats to human health in the past. Other human pathogenic coronaviruses have been identified, and they are known to cause respiratory diseases with manifestations ranging from mild to severe. In this study, we evaluated the performance of a multiplex RT-rPCR specific to seven human pathogenic coronaviruses in mainly detecting SARS-CoV-2 directly from nasopharyngeal swabs obtained from suspected COVID-19 infected patients, while simultaneously detecting different human pathogenic coronaviruses in case these were also present. We tested 1195 clinical samples suspected of COVID-19 infection. The assay identified that 69% of the samples tested positive for SARS-CoV-2 (1195), which was confirmed using another SARS-CoV-2 RT-PCR kit available in our laboratory. None of these clinical samples were positive for SARS-CoV, MERS-CoV or HCoV. This means that during the endemic phase of COVID-19, infection with other human pathogenic coronaviruses, even the common cold coronavirus (HCoV), is very uncommon. Our study also confirmed that the multiplex RT-rPCR is a sensitive assay for detecting SARS-CoV-2 regardless of differences among the variants. This multiplex RT-rPCR is also time- and cost-saving and very easy to apply in the diagnostic laboratory due to its simple procedure and its stability in storage after preparation. These features make the assay a valuable approach in screening procedures for the rapid detection of SARS-CoV-2 and other human pathogenic coronaviruses that could affect public health.

Keywords: COVID-19; SARS-CoV; SARS-CoV-2; antispike; diagnostic accuracy; human pathogenic coronavirus; multiplex RT-rPCR; vaccines.

Figure 1

Shows the input of RNA extracted from the sample, the PC and the NC into the MPL1 and MPL2 to run the MPL RT-rPCR. The PC and NC were added to the MPL1 and MPL2 for each run of the assay, not for each sample.

Figure 2

Example of amplification curve for a positive SARS-CoV-2 sample. Multiple reaction curves indicate amplification of the positive clinical sample using two multiplex RT-rPCR master mixes. N1–3: SARS-CoV-2 nucleocapsid gene; RP: RNaseP gene; E: SARS-CoV-2 envelope gene; PEDV: porcine epidemic diarrhea virus.

Figure 3

Violin plot of the cycle threshold (Ct) value of multiplex RT-rPCR. The violin plot shows the cycle threshold (Ct) values obtained for each positive SARS-CoV-2 sample tested (n = 835). For each plot, the continuous line indicates the median value: E = 20; N1 = 19; N2 = 18; N3 = 19; RP = 28; PEDV = 28. No amplification curve was obtained for HCoV-HKU-1-PR or upE_TqPR. E = envelope gene; N1-N3 = nucleocapsid gene; RP = RNaseP gene; PEDV = porcine epidemic diarrhea virus. The data were plotted using GraphPad Prism 9.3.1.

Figure 4

Comparison of cycle threshold (Ct) values of two multiplex RT-rPCR sessions performed at different times. The violin plot shows the cycle threshold (Ct) values obtained during two different assays performed on 321 positive SARS-CoV-2 samples at time zero (Assay 1) and after six months (Assay 2). The plot shows that the Ct value is very similar between the two independently performed assays. This result confirmed the stability of multiplex RT-rPCR master mix conferred by Enzyme Stabilizer. The continuous line inside the plot indicates the median value. Assay 1: E = 21; N1 = 20; N2 = 19; N3 = 20; RP = 28; PEDV = 28. Assay 2: E = 21; N1 = 20; N2 = 19; N3 = 20; RP = 28; PEDV = 28. No significant difference (p < 0.05) was revealed by t-test. E = envelope gene; N1–N3 = nucleocapsid gene; RP = RNaseP gene; PEDV = porcine epidemic diarrhea virus. The data were plotted using GraphPad Prism 9.3.1.