Introduction
Material and method
Samples recruitment
Sample preparation
Pooled sample and serial dilution
Viral genome extraction and RT-qPCR
Gene | Primer | Primer length (bp) | Product length |
---|---|---|---|
S | F 5ʹ-TCCATCAAAACCAAGCAAGA-3ʹ | 20 | 245 bp |
R 5ʹ-CACCAAAGGTCCAACCAGAA-3ʹ | 20 | NC_045512v2:23,986 + 24,230 | |
N | F 5ʹ-CTGGACTTCCCTATGGTGCT-3ʹ | 20 | 284 bp |
R 5ʹ-ATTGCCAGCCATTCTAGCAG-3ʹ | 20 | NC_045512v2:28,629 + 28,912 |
Determination of analytical specificity
Static analysis
Results
Subjects and specimens
Data | Finding | Total n = 210 | Positive sample n = 110 | Negative sample n = 100 | P value |
---|---|---|---|---|---|
n (%) | n (%) | n (%) | |||
Gender | Male | 52.00% | 53.50% | 50.40% | 0.083 |
Female | 48.00% | 46.50% | 49.60% | ||
Age (years) | 11–40 | 60.50% | 59.80% | 61.30% | |
41–70 | 35.50% | 36.20% | 34.70% | 0.076 | |
70< | 3.90% | 3.90% | 3.90% | ||
Location | City | 53.90% | 70.10% | 37.80% | – |
Village | 46.10% | 29.90% | 62.20% | ||
Lung disease | Yes | 1.60% | 3.10% | 0.00% | – |
No | 98.40% | 96.90% | 100.00% | ||
Fever | Yes | 9.10% | 9.40% | 8.70% | 0.158 |
No | 90.90% | 90.60% | 91.30% | ||
Sore throat | Yes | 25.60% | 31.50% | 19.70% | 0.063 |
No | 74.40% | 68.50% | 80.30% | ||
Headache | Yes | 27.20% | 28.30% | 26% | 0.118 |
No | 72.80% | 71.70% | 74% | ||
Cough | Yes | 20.50% | 24.40% | 16.50% | 0.068 |
No | 79.50% | 75.60% | 83.50% |
Standard dilution | Copies/ml | One step RT-qPCR RdRp gene | One-step RT-qPCR N gene | Two step RT-qPCR S gene | Two step RT-qPCR N gene | ||
---|---|---|---|---|---|---|---|
Ct (Threshold 2) | Ct (Threshold 2) | \(\mathrm{Ct }(\mathrm{Threshold }0.2 )\) | Tm (°C) | \(\mathrm{Ct }(\mathrm{Threshold }0.2 )\) | Tm (°C) | ||
0.1 | 109 | 19.97 | 16.44 | 18.56 | 83 | 19.93 | 85 |
0.01 | 108 | 21.49 | 19.14 | 24.13 | 83 | 25.49 | 85 |
0.001 | 107 | 24.03 | 21.56 | 27.52 | 83 | 28.14 | 85 |
0.0001 | 106 | 27.5 | 23.82 | 32.06 | 83 | 32.31 | 85 |
0.00001 | 105 | 31.56 | 27.31 | 36.05 | 83 | 36.91 | 85 |
0.000001 | 104 | 34.66 | 30.77 | 39.41 | 83 | 39.18 | 85.14 |
0.0000001 | 103 | 36.74 | 33.23 | 41.08 | 83.19 | N.D | – |
0.00000001 | 102 | 40.01 | 37.83 | N.D | – | N.D | – |
Analytical specificity of two-step RT-qPCR techniques
RT-qPCR techniques validation in clinical samples
Method | Target | Accuracy (%) | PPV (%) | NPV (%) |
---|---|---|---|---|
Two step RT-qPCR (SYBR Green-based) | S gene | 88 | 85 | 91 |
N gene | 86 | 84 | 87 | |
One step RT-qPCR (Prob-based) | RdRp gene | 86 | 82 | 90 |
N gene | 94 | 92 | 95 |
Visualization of RT-qPCR amplicon
ROC curve analysis
Target | Method | AUC (95% CI) | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) | Cut-off value | P-value |
---|---|---|---|---|---|---|
S gene | Two step RT-qPCR | 0.90 (0.79–0.97) | 88 (68.8–97.5) | 88 (68.8–97.5) | ≤ 40 | 0.0471 |
N gene | Two step RT-qPCR | 0.79 (0.66–0.89) | 88 (68.8–97.5) | 84 (63.9–95.5) | ≤ 40 | 0.0489 |
RdRp gene | One step RT-qPCR | 0.95 (0.86–0.99) | 92 (74.0–99) | 80 (59.3–93.2) | ≤ 40 | 0.0234 |
N gene | One step RT-qPCR | 0.95 (0.86–0.99) | 92 (74.0–99) | 96 (79.6–99.9) | ≤ 40 | 0.0356 |
S and N gene | Two step RT-qPCR | 0.85 (0.72–0.93) | 88 (68.8–97.5) | 86 (66.3–96.5) | ≤ 40 | 0.0461 |
RdRp and N gene | One step RT-qPCR | 0.95 (0.86–0.99) | 92 (74.45–99.8) | 88 (69.4–96.55) | ≤ 40 | 0.0441 |