Fig 1.
Proteinase K improves the performance of the heat inactivation method in RT-qPCR determinations of SARS-CoV-2.
(a-b) Positive (#8, #11, #12, #16, #19, #20) and negative (#1, #2, #3) nasopharyngeal swab samples were processed by heat inactivation (HID, 98°C for 5 min); proteinase K treatment followed by heat inactivation (PK+HID, 55°C for 15 min and 98°C for 5 min) or subjected to RNA extraction (purified RNA). The viral N1 and N2 genes and the human RNase P gene (RP) were amplified and detected by RT-qPCR. (a) CT values obtained from RT-qPCR analysis of the same samples prepared by the three different methods. (b) Representative amplification curves for each gene obtained for one of the positive samples. (c) Amplification efficiencies (EPCR) and initial amount of amplicon copies (n) in PK+HID samples relative to the corresponding HID samples. The median of each measurement is represented with a line in the bars and the lengths of these bars represent the standard error. (d) Positive nasopharyngeal swab samples were subjected to treatment with different concentrations of proteinase K (PK) followed by heat inactivation (55°C for 15 min and 98°C for 5 min). The viral N1 and N2 genes and the human RP gene were amplified and detected by RT-qPCR. ΔCT represents the mean difference between CT values obtained in each analyzed condition and the corresponding to HID samples. Mean ± SEM values are represented (N = 5).
Fig 2.
PK+HID method exhibits a similar performance than RNA extraction in RT-qPCR determinations of the SARS-CoV-2 N1 gene.
Positive nasopharyngeal swab samples were processed for RNA extraction (purified RNA) or subjected to treatment with PK 1 mg/ml (55°C for 15 min) followed by heat inactivation at 98°C for 5 min (PK+HID). The viral N1 gene and the human RP gene were amplified and detected by RT-qPCR. CT values obtained for the same samples prepared by the two different methods (N = 27), the line obtained by regression of the data (continuous lines) and 95% confidence bands (pink) are represented. The parameter values obtained from the fitting were: slope = 1.17 ± 0.05 and intercept = -3.4 ± 1.2 (N1 amplicon); slope = 1.0 ± 0.1 and intercept = 1.7 ± 3.0 (RP amplicon).
Fig 3.
PK+HID method exhibits a similar performance than RNA extraction in RT-qPCR determinations of the SARS-CoV-2 N and ORF1ab genes.
Positive nasopharyngeal swab samples were processed for RNA extraction (purified RNA) or subjected to treatment with PK 1 mg/ml (55°C for 15 min) followed by heat-inactivation at 98°C for 5 min (PK+HID). The viral N and ORF1ab genes and the human RP gene were amplified and detected by RT-qPCR. (a) CT values obtained for the same samples prepared by the two different methods, the line obtained by regression of the data (continuous lines) and 95% confidence bands (pink) are represented. The parameter values obtained from the fitting were: slope = 1.02 ± 0.04 and intercept = 0.1 ± 0.9 (N amplicon); slope = 1.02 ± 0.04 and intercept = 0.8 ± 0.9 (ORF1ab amplicon) and slope = 0.4 ± 0.2 and intercept = 15 ± 6 (RP amplicon). Notice that CT values determined for RP spans a smaller range. (b) Amplification efficiencies obtained in PK+HID samples (EPK+HID) relative to the corresponding purified RNA samples (Epurified RNA). The mean values obtained for Epurified RNA were: 1.98 ± 0.03 (N), 2.02 ± 0.06 (ORF1ab) and 1.94 ± 0.03 (RP). The median of each measurement is represented with a line in the bars and the lengths of these bars represent the standard error (N = 16).
Fig 4.
Performance of PK+HID method in RT-qPCR determinations of the SARS-CoV-2 N, RdRp and E genes in randomly-selected clinical specimens.
Nasopharyngeal swab samples were processed for RNA extraction (purified RNA) or subjected to treatment with PK 1 mg/ml (55°C for 15 min) followed by heat-inactivation at 98°C for 5 min (PK+HID). The viral N, E and, RdRp genes and the human RP gene were amplified and detected by RT-qPCR. (a) CT values obtained for the same samples prepared by the two different methods, the line obtained by regression of the data (continuous lines) and 95% confidence bands (pink) are represented. RP plot also includes the data obtained in SARS-CoV-2 negative samples and spans a smaller CT range. The parameter values obtained from the fitting were: slope = 1.2 ± 0.1 and intercept = -5 ± 3 (N amplicon); slope = 0.7 ± 0.2 and intercept = 9 ±4 (RdRp amplicon); slope = 0.7 ± 0.1 and intercept = 7 ± 3 (E amplicon) and slope = 0.4 ± 0.1 and intercept = 16 ± 3 (RP amplicon). (b) Amplification efficiencies obtained in PK+HID samples (EPK+HID) relative to the corresponding purified RNA samples (Epurified RNA). The mean values obtained for Epurified RNA were: 1.94 ± 0.02 (N), 1.97 ± 0.02 (RdRp), 1.93 ± 0.03 (E) and 1.90 ± 0.03 (RP). The median of each measurement is represented with a line in the bars and the lengths of these bars represent the standard error (N = 8).