Background
Methods
Viruses
Materials
Selection of references
Positive references
Negative references
Preparation of the references
Confirmation of the references
Sequence confirmation of the references
Confirmation by quantitative PCR (q-PCR)
Confirmation of sensitivity
Confirmation of accuracy
Stability of the references
Collaborative calibration of the references
Statistical analysis
Results
Preparation of the references
Specificity of the references
Strains | Ct value | |||||
---|---|---|---|---|---|---|
Specific primers and probe | SinoMD kit | DaAn kit | ||||
Exp 1 | Exp 2 | Exp 1 | Exp 2 | Exp 1 | Exp 2 | |
AH12 | 22.89 | 22.84 | 24.69 | 26.50 | 20.72 | 18.60 |
HN1 | 24.05 | 23.53 | 24.96 | 26.04 | 20.91 | 20.15 |
JS3 | 25.01 | 25.26 | 25.01 | 25.50 | 21.94 | 20.84 |
LN3 | 24.29 | 23.96 | 24.26 | 22.61 | 21.26 | 20.04 |
SD4 | 24.09 | 24.05 | 26.90 | 29.86 | 21.42 | 19.27 |
HB29 | 23.68 | 22.99 | 25.30 | 23.12 | 20.44 | 18.87 |
Positive | 21.89 | 21.57 | 17.29 | 18.99 | 23.49 | 19.77 |
Negative | — | — | — | — | — | — |
Sensitivity of the references
HB29 strain | Ct value | |||||||
---|---|---|---|---|---|---|---|---|
SinoMD kit | DaAn kit | |||||||
Exp 1 | Exp 2 | Exp 3 | Exp 4 | Exp 1 | Exp 2 | Exp 3 | Exp 4 | |
S1 (105 U/mL) | 30.03 | 27.35 | 29.40 | 30.23 | 21.42 | 22.48 | 26.37 | 24.11 |
S2 (104 U/mL) | 32.21 | 33.16 | 32.74 | 33.27 | 26.05 | 24.84 | 28.86 | 26.69 |
S3 (103 U/mL) | 37.07 | 34.04 | 35.60 | 36.19 | 28.28 | 28.32 | 30.75 | 28.13 |
S4 (102 U/mL) | — | 37.36 | — | — | 32.20 | 29.46 | 31.79 | 31.08 |
S5 (10 U/mL) | — | — | — | — | — | 32.37 | 32.06 | 31.17 |
Positive | 17.59 | 16.75 | 17.84 | 19.07 | 19.77 | 23.49 | 20.69 | 20.00 |
Negative | — | — | — | — | — | — | — | — |
Repeatability of the references
References | Ct value | |||
---|---|---|---|---|
SinoMD kit | DaAn kit | |||
Exp 1 | Exp 2 | Exp 1 | Exp 2 | |
R1 | 33.27 | 33.80 | 26.05 | 27.47 |
R2 | 34.19 | 34.98 | 27.63 | 27.08 |
R3 | 30.72 | 33.10 | 27.67 | 26.22 |
R4 | 31.24 | 33.03 | 27.38 | 28.15 |
R5 | 32.75 | 36.36 | 26.26 | 27.12 |
R6 | 32.19 | 34.00 | 26.14 | 25.56 |
R7 | 31.85 | 33.34 | 26.11 | 25.67 |
R8 | 32.62 | 33.93 | 25.29 | 24.84 |
R9 | 34.43 | 32.74 | 27.34 | 28.03 |
R10 | 30.90 | 34.18 | 26.89 | 25.09 |
SD | 1.29 | 1.07 | 0.81 | 1.21 |
Average | 32.42 | 33.95 | 26.68 | 26.52 |
CV (%) | 3.98 | 3.16 | 3.05 | 4.55 |
Stability of all references
References | Ct value | Normal value | |||
---|---|---|---|---|---|
37 °C for 8 days | Room temperature for 14 days | −70 °C for 8 months | |||
Positive | P1 (AH12) | 18.02 | 22.81 | 22.84 | 18.60 |
P2 (HN1) | 18.49 | 24.02 | 23.53 | 20.15 | |
P3 (JS3) | 24.83 | 25.07 | 25.26 | 20.84 | |
P4 (LN3) | 18.80 | 24.52 | 23.96 | 20.04 | |
P5 (SD4) | 22.41 | 23.88 | 24.05 | 19.27 | |
P6 (HB29) | 17.92 | 22.35 | 22.99 | 18.87 | |
Sensitivity | S1 (105U) | 20.20 | 26.69 | 25.51 | 21.42 |
S2 (104U) | 23.99 | 30.03 | 27.46 | 26.05 | |
S3 (103U) | 27.40 | 32.23 | 30.78 | 28.28 | |
S4 (102U) | 35.29 | 35.54 | 31.08 | 32.20 | |
S5 (10U) | — | 37.23 | 32.68 | — | |
Accuracy | R1 | 27.50 | 29.87 | 27.77 | 27.47 |
R2 | 24.56 | 29.78 | 28.08 | 27.08 | |
R3 | 27.50 | 29.21 | 27.89 | 26.22 | |
R4 | 24.30 | 30.61 | 27.89 | 28.15 | |
R5 | 24.27 | 30.04 | 27.63 | 27.12 | |
R6 | 23.99 | 28.70 | 27.85 | 25.56 | |
R7 | 25.09 | 30.03 | 27.81 | 25.67 | |
R8 | 25.11 | 31.76 | 28.01 | 24.84 | |
R9 | 25.01 | 31.39 | 27.60 | 28.03 | |
R10 | 25.06 | 31.16 | 27.37 | 25.09 | |
SD | 1.26 | 0.97 | 0.21 | 1.21 | |
Average | 25.24 | 30.26 | 27.79 | 26.52 | |
CV | 5.00% | 3.20% | 0.75% | 4.55% |
Reference | Ct value | ||
---|---|---|---|
First F-T | Second F-T | Third F-T | |
HB29 strain | 25.49 | 26.23 | 27.31 |
26.29 | 25.80 | 26.58 | |
26.10 | 26.28 | 25.80 | |
Average | 25.96 | 26.10 | 26.56 |
HB29 strain at 1:100 | 33.27 | 31.85 | 34.43 |
34.19 | 32.62 | 30.90 | |
30.72 | 32.75 | 33.48 | |
31.24 | 32.19 | 33.00 | |
Average | 32.36 | 32.35 | 32.95 |
Calibration of the references
Labs | Exp | Positive coincidence rate | Negative coincidence rate | Sensitivity (102 U/mL) | Accuracy (CV) |
---|---|---|---|---|---|
Second group for in vitro diagnostics of NIFDC | 1 | 100% | 100% | Detected | 3.49% |
2 | 100% | 100% | Detected | 0.45% | |
3 | 100% | 100% | Detected | 3.80% | |
Average | 100% | 100% | Detected | 2.58% | |
Arbovirus vaccine group of NIFDC | 1 | 100% | 100% | Detected | 3.98% |
2 | 100% | 100% | Detected | 3.16% | |
3 | 100% | 100% | Detected | 3.05% | |
4 | 100% | 100% | Detected | 4.55% | |
Average | 100% | 100% | Detected | 3.69% | |
DaAn gene Co. Ltd | 1 | 100% | 100% | Detected | 1.97% |
2 | 100% | 100% | Detected | 1.53% | |
3 | 100% | 100% | Detected | 0.56% | |
4 | 100% | 100% | Detected | 1.44% | |
5 | 100% | 100% | Detected | 1.19% | |
Average | 100% | 100% | Detected | 1.34% |