Reagent | Manufacturer: |
HIV-1M Secondary Standard, 50′000 | Roche |
cp/ML | |
HBV Secondary Standard, 400 IU/ml | Roche |
CT (DNA POS CTL pCHL-1) | Roche |
Instrument | Manufacturer |
Hamilton Star | Hamilton Medical AG |
(Bonaduz, CH) | |
Light Cycler 480 | Roche Diagnostics GmbH |
(Mannheim, DE) | |
Chameleon Sealer | K biosystems (Essex, UK) |
Compressor | K biosystems (Essex, UK) |
Reagent | Manufacturer: |
PreservCyt | Thin Prep |
K3 EDTA Plasma, PCR neg. | Roche |
Target | Concentration | Matrix | |
HBV | 50 | IU/ml | K3 EDTA plasma |
HIV-1M | 100 | cp/ml | K3 EDTA plasma |
CT | 2.5 | fg/ml | PreservCyt |
IC/IQS—Storage Buffer | Conc. or pH |
Tris (mM) | 10 |
EDTA (mM) | 0.1 |
Sodium Azide (w/v, %) | 0.05 |
Poly rA RNA (mg/l) | 20 |
pH | 8 |
Conc. or pH | |
Protease reagent | |
Tris (mM) | 10 |
EDTA (mM) | 1 |
Calcium Chloride (mM) | 5 |
Calcium Acetate (mM) | 5 |
Esperase (mg/ml) | 80 |
Glycerin (w/v, %) | 50 |
pH | 5.5 |
MGP Reagent | |
MPG Powder (mg/ml) | 60 |
Tris (mM) | 30 |
Methylparaben (w/v, %) | 0.1 |
Sodium Azide (w/v, %) | 0.095 |
pH | 8.5 |
Lysis Reagent | |
Guanidine Thiocyanate (M) | 4 |
Sodium Citrate (mM) | 50 |
Polydocanol (w/v, %) | 5 |
Dithiotreitol (w/v, %) | 2 |
pH | 5.8 |
Wash buffer | |
Sodium Citrate (mM) | 7.5 |
Methylparaben (w/v, %) | 0.1 |
pH | 4.1 |
Elution buffer | |
Tris (mM) | 30 |
Methylparaben (w/v, %) | 0.2 |
pH | 8.5 |
Concentration/50 | |
μl-PCR [μM] | |
R1 Reagent | |
Water (PCR grade) | |
Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic | 3′000 |
Acid) | |
NaN3/Ri, buffered with 10 mM Tris at pH 7 [%] | 0.018 |
R2 Reagent | |
DMSO [%] | 5.000% |
NaN3/Ri, buffered with 10 mM Tris at pH 7 [%] | 0.027% |
Potassium acetate pH 7.0 | 110′000 |
Glycerol [%] | 3.000% |
Tricine pH 8.0 | 50′000 |
Igepal [%] | 0.024% |
dGTP | 337.5 |
dATP | 337.5 |
dCTP | 337.5 |
dUTP | 675 |
Primers/probes selected from SEQ ID NOs 1-35 | 0.1-0.15 |
SEQ ID NO 42 | 0.1 |
SEQ ID NO 43 | 0.1 |
SEQ ID NO 44 | 0.1 |
Uracil-N-Glycosylase | 10 (U/reaction) |
Z05-D Polymerase | 40 (U/reactionn) |
NTQ21-46A—Aptamer | 0.222 |
Water |
Concentration/50 | ||
μl-PCR | ||
R2 Reagent | ||
H20 | 100 | % |
Tricine 7.7 | 40 | mM |
Tween | 0.03 | % (v/v) |
Glycerol | 5 | % (v/v) |
KOH | 25.2 | mM |
KOAc | 121.8 | mM |
NTQ21-46A (Aptamer) | 0.2625 | uM |
dGTP | 0.42 | uM |
dATP | 0.42 | uM |
dCTP | 0.42 | uM |
dUTP | 0.84 | uM |
SEQ ID NO 36 | 1.2 | uM |
SEQ ID NO 37 | 0.1 | uM |
SEQ ID NO 38 | 1.2 | uM |
SEQ ID NO 42 | 0.6 | uM |
SEQ ID NO 43 | 0.6 | uM |
SEQ ID NO 44 | 0.15 | uM |
Z05D Polymerase | 35 | (U/reaction) |
Uracil-N-Glycosylase | 2 | (U/reaction) |
Sodium Azide | 0.027 | % (m/v) |
R1 Reagent | ||
H20 | 100 | % |
MgOAc | 2.5 | mM |
MnOAc pH 6.1 | 2.5 | mM |
Sodium Azide | 0.018 | % (m/v) |
Concentration/50 | ||
μl-PCR | ||
R1 Reagent | ||
Water (PCR grade) | ||
Mn(Ac)2 (pH 6.5 in 0.002% (V/V) Glacial Acetic | 2.7 | mM |
Acid) | ||
NaN3 | 0.0135% | (W/V) |
R2 Reagent | ||
NaN3/Ri, buffered with 10 mM Tris at pH 7 [%] | 0.0315% | |
Potassium acetate | 112.4 | mM |
Glycerol [%] | 3.5% | |
Tricine | 61 | mM |
Potassium hydroxide | 28.4 | mM |
dGTP | 525 | uM |
dATP | 525 | uM |
dCTP | 525 | uM |
dUTP | 1.05 | mM |
SEQ ID NO 39 | 750 | nM |
SEQ ID NO 40 | 600 | nM |
SEQ ID NO 41 | 116 | nM |
Aptamer NTQ-46A | 175 | nM |
Uracil-N-Glycosylase | 5 U/reaction | |
Z05-D Polymerase | 31 U/reaction |
Thermo cycling profile | ||||||
Program | Target | Acquisition | Hold | Ramp Rate | Analysis | |
Name | (° C.) | Mode | (hh:mm:ss) | (° C./s) | Cycles | Mode |
Pre-PCR | 50 | None | 00:02:00 | 4.4 | 1 | None |
94 | None | 00:00:05 | 4.4 | |||
55 | None | 00:02:00 | 2.2 | |||
60 | None | 00:06:00 | 4.4 | |||
65 | None | 00:04:00 | 4.4 | |||
1st | 95 | None | 00:00:05 | 4.4 | 5 | Quantification |
Measurement | 55 | Single | 00:00:30 | 2.2 | ||
2nd | 91 | None | 00:00:05 | 4.4 | 45 | Quantification |
Measurement | 58 | Single | 00:00:25 | 2.2 | ||
Cooling | 40 | None | 00:02:00 | 2.2 | 1 | None |
Detection Format (Manual) | |
Filter Combination | Integration Time (sec) |
435-470 | 1 |
495-525 | 0.5 |
540-580 | 0.5 |
610-645 | 0.5 |
680-700 | 1 |
Target | Acquisition | Plateau | Measure | Ramp Rate | ||
[° C.] | Mode | [hh:mm:ss] | [hh:mm:ss] | [° C./s] | ||
Pre- | UNG-Step | 50 | none | 00:02:00 | 00:00:00 | 2.2 |
PCR | UNG/Template | 94 | none | 00:00:05 | 00:00:00 | 4.4 |
Denaturation | ||||||
RT-Step | 55 | none | 00:02:00 | 00:00:00 | 2.2 | |
60 | none | 00:06:00 | 00:00:00 | 4.4 | ||
65 | none | 00:04:00 | 00:00:00 | 4.4 | ||
1st Measurement | 95 | none | 00:00:05 | 00:00:00 | 4.4 | |
55 | single | 00:00:30 | 00:00:08 | 2.2 | ||
2nd Measurement | 91 | none | 00:00:05 | 00:00:00 | 4.4 | |
58 | single | 00:00:25 | 00:00:08 | 2.2 | ||
Cooling | 40 | none | 00:02:00 | 00:00:00 | 2.2 |
Name | Cycles |
Pre-PCR | 1 |
1st Measurement | 5 |
2nd Measurement | 45 |
Cooling | 1 |
Conc. in 50 | |
ul-PCR (uM) | |
Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) | 3′300 |
NaN3/Ri, buffered with 10 mM Tris at pH 7 | 0.018 |
pH: 6.41 |
Conc. in 50 | |
Reagent | ul-PCR (uM) |
DMSO (%) | 5.4 |
NaN3/Ri, buffered with 10 mM Tris at pH 7 | 0.027 |
KOAc (pH 7.0) | 120′000 |
Glycerol (%) | 3 |
Tween 20 (%) | 0.015 |
Tricine pH 8.0 | 60′000 |
NTQ21-46A—Aptamer | 0.2222 |
Uracil-N-Glycosylase (U/uL) | 0.2 |
dGTP | 400.0 |
dATP | 400.0 |
dCTP | 400.0 |
dUTP | 800.0 |
ZO5-D Polymerase (U/ul)* | 0.9 |
Primers/probes selected from SEQ ID NOs 1-35 | 0.125-0.3 |
SEQ ID NO 36 | 0.100 |
SEQ ID NO 37 | 0.100 |
SEQ ID NO 38 | 0.150 |
Primers/probes selected from SEQ ID NOs 60-76 | 0.050-0.250 |
SEQ ID NO 42 | 0.200 |
SEQ ID NO 43 | 0.200 |
SEQ ID NO 44 | 0.100 |
TABLE 1 | |||
HIV-1 Group M Hit rates and Probit LOD from individual panel | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
32 cp/mL | 21 | 21 | 100% |
16 cp/mL | 21 | 21 | 100% |
8 cp/mL | 21 | 21 | 100% |
4 cp/mL | 21 | 20 | 95% |
2 cp/mL | 21 | 15 | 71% |
1 cp/mL | 21 | 9 | 43% |
0 cp/mL (neg. control) | 12 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 4.06 cp/mL | ||
95% confidence interval for LOD by PROBIT | 2.85-9.24 | ||
analysis | cp/mL |
TABLE 2 | |||
HIV-1 Group O Hit rates and Probit LOD from individual panel | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
60 cp/mL | 21 | 21 | 100% |
30 cp/mL | 20 | 20 | 100% |
20 cp/mL | 21 | 21 | 100% |
14 cp/mL | 21 | 19 | 90% |
7 cp/mL | 21 | 15 | 71% |
4.5 cp/mL | 21 | 12 | 57% |
0 cp/mL (neg. control) | 12 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 14.9 cp/mL | ||
95% confidence interval for LOD by PROBIT | 10.9-31.5 | ||
analysis | cp/mL |
TABLE 3 | |||
HIV-2 Hit rates and Probit LOD from individual panel | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
4 cp/mL | 21 | 21 | 100% |
2 cp/mL | 21 | 21 | 100% |
1 cp/mL | 21 | 20 | 95% |
0.5 cp/mL | 21 | 13 | 62% |
0.25 cp/mL | 21 | 13 | 62% |
0.125 cp/mL | 21 | 7 | 33% |
0 cp/mL (neg. control) | 12 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 1.29 cp/mL | ||
95% confidence interval for LOD by PROBIT | −3.11 cp/mL | ||
analysis |
TABLE 4 | |||
HBV Hit rates and Probit LOD from individual panel | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
7.6 IU/mL | 21 | 21 | 100% |
3.8 IU/mL | 21 | 21 | 100% |
1.9 IU/mL | 21 | 20 | 95% |
0.95 IU/mL | 21 | 14 | 67% |
0.6 IU/mL | 19 | 12 | 63% |
0.4 IU/mL | 21 | 12 | 57% |
0 IU/mL (neg. control) | 12 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 2.27 IU/mL | ||
95% confidence interval for LOD by PROBIT | 1.48-6.54 | ||
analysis | IU/mL |
TABLE 5 | |||
HCV Hit rates and Probit LOD from individual panel | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
24 IU/mL | 21 | 21 | 100% |
12 IU/mL | 21 | 21 | 100% |
6 IU/mL | 21 | 21 | 100% |
3 IU/mL | 21 | 17 | 81% |
1.5 IU/mL | 21 | 14 | 67% |
0.75 IU/mL | 21 | 9 | 43% |
0 IU/mL (neg. control) | 18 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 4.76 IU/mL | ||
95% confidence interval for LOD by PROBIT | 3.14-11.61 | ||
analysis | IU/mL |
Conc. in 50 | |
Reagent | ul-PCR (uM) |
Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) | 3′300 |
NaN3/Ri, buffered with 10 mM Tris at pH 7 | 0.018 |
pH: 6.41 |
Conc. in 50 | |
Reagent | ul-PCR (uM) |
DMSO (%) | 5.4 |
NaN3/Ri, buffered with 10 mM Tris at pH 7 | 0.027 |
K acetate pH 7.0 | 120′000 |
Glycerol (%) | 3 |
Tween 20 (%) | 0.015 |
Tricine pH 8.0 | 60′000 |
NTQ21-46A—Aptamer | 0.2222 |
Uracil-N-Glycosylase (U/uL) | 0.2 |
dGTP | 400.0 |
dATP | 400.0 |
dCTP | 400.0 |
dUTP | 800.0 |
ZO5-D Polymerase (U/ul)* | 0.9 |
Primers/probes selected from SEQ ID NOs 53-59 | 0.08-0.4 |
SEQ ID NO 42 | 0.150 |
SEQ ID NO 43 | 0.150 |
SEQ ID NO 44 | 0.100 |
TABLE 6 | |||
WNV Hit rates and Probit LOD from individual panel | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
20 cp/mL | 21 | 21 | 100% |
12 cp/mL | 21 | 21 | 100% |
8 cp/mL | 21 | 21 | 100% |
5 cp/mL | 21 | 17 | 81% |
2.5 cp/mL | 21 | 15 | 71.4% |
0.5 cp/mL | 21 | 1 | 4.8% |
0 cp/mL (neg. control) | 12 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 6.57 cp/mL | ||
95% confidence interval for LOD by PROBIT | 4.74-11.03 | ||
analysis | cp/mL |
TABLE 7 | |||
SLEV Hit rates and Probit LOD from individual panel | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
140 cp/mL | 21 | 21 | 100% |
100 cp/mL | 21 | 20 | 95.2% |
70 cp/mL | 21 | 20 | 95.2% |
40 cp/mL | 21 | 17 | 81.0% |
20 cp/mL | 21 | 11 | 52.4% |
10 cp/mL | 21 | 6 | 28.6% |
0 cp/mL (neg. control) | 12 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 78.9 cp/mL | ||
95% confidence interval for LOD by PROBIT | 55.4-145.7 | ||
analysis | cp/mL |
TABLE 8 | |||
JEV Hit rates and Probit LOD from individual panel | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
20 cp/mL | 21 | 20 | 95.2% |
12 cp/mL | 21 | 20 | 95.2% |
8 cp/mL | 21 | 18 | 85.7% |
5 cp/mL | 21 | 17 | 81.0% |
2.5 cp/mL | 21 | 14 | 66.7% |
0.5 cp/mL | 21 | 2 | 9.52% |
0 cp/mL (neg. control) | 12 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 13.55 cp/mL | ||
95% confidence interval for LOD by PROBIT | 8.78-27.7 | ||
analysis | cp/mL |
Final Conc. in 50 | |
Reagent | ul-PCR (uM) |
Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) | 3′300 |
NaN3/Ri, buffered with 10 mM Tris at pH 7 | 0.018 |
pH: 6.41 |
Final Conc. in 50 | ||
Reagent | ul-PCR (uM) | |
Glycerol (%, w/v) | 3% | |
Tricine | 60 | mM |
DMSO (%, v/v) | 5.4% | |
KOAc | 120 | mM |
Tween 20 (v/v) | 0.015% | |
Aptamer NTQ21-46 A | 0.222 | μM |
ZO5D Polymerase | 0.9 U/μL (45 U/rxn) | |
Uracil-N-Glycosylase | 0.2 U/μL (10 U/rxn) | |
Sodium Azide (w/v) | 0.027% | |
dCTPs | 400 | μM |
dGTPs | 400 | μM |
dATPs | 400 | μM |
dUTPs | 800 | μM |
SEQ ID NO 36 | 1.2 | μM |
SEQ ID NO 37 | 1.2 | μM |
SEQ ID NO 50 | 0.6 | μM |
SEQ ID NO 51 | 0.6 | μM |
SEQ ID NO 38 | 0.1 | μM |
SEQ ID NO 52 | M |
TABLE 9 | |||
LOD analysis for 200 μL input volume in EDTA-plasma. * | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
25 IU/mL | 41 | 41 | 100% |
15 IU/mL | 41 | 39 | 95.1% |
10 IU/mL | 41 | 40 | 97.6% |
7 IU/mL | 41 | 40 | 97.6% |
4 IU/mL | 24 | 20 | 83.3% |
1 IU/mL | 24 | 4 | 16.7% |
0 IU/mL (neg. control) | 24 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 8.2 IU/mL | ||
95% confidence interval for LOD by PROBIT | 4.8-26.0 | ||
analysis | IU/mL | ||
* Additional replicates were tested to narrow the observed 95% confidence interval. |
TABLE 10 | |||
LOD analysis for 500 μL input volume in EDTA-plasma. | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
10 IU/mL | 21 | 21 | 100% |
7 IU/mL | 21 | 21 | 100% |
4 IU/mL | 21 | 21 | 100% |
2.5 IU/mL | 21 | 20 | 95.2% |
1 IU/mL | 21 | 14 | 66.7% |
0.2 IU/mL | 21 | 1 | 4.8% |
0 IU/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 2.3 IU/mL | ||
95% confidence interval for LOD by PROBIT | 1.6-4.2 | ||
analysis | IU/mL |
TABLE 11 | |||
LOD analysis for 200 μL input volume in serum. | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
25 IU/mL | 21 | 21 | 100% |
15 IU/mL | 21 | 20 | 95.2% |
10 IU/mL | 21 | 21 | 100% |
7 IU/mL | 21 | 20 | 95.2% |
4 IU/mL | 21 | 15 | 71.4% |
1 IU/mL | 21 | 8 | 38.1% |
0 IU/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 9.4 IU/mL | ||
95% confidence interval for LOD by PROBIT | 6.2-19.0 | ||
analysis | IU/mL |
TABLE 12 | |||
LOD analysis for 500 μL input volume in serum. | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
10 IU/mL | 21 | 21 | 100% |
7 IU/mL | 21 | 21 | 100% |
4 IU/mL | 21 | 21 | 100% |
2.5 IU/mL | 21 | 16 | 76.2% |
1 IU/mL | 21 | 16 | 76.2% |
0.2 IU/mL | 21 | 7 | 33.3% |
0 IU/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 4.1 IU/mL | ||
95% confidence interval for LOD by PROBIT | 2.4-10.0 | ||
analysis | IU/mL |
TABLE 13 | ||||
Linearity in EDTA Plasma | ||||
Assigned | Mean Log 10 | |||
Nominal Titer | Assigned Titer | Log10 | Titer | Repli- |
(IU/mL) | (IU/mL) | Titer | observed | cates |
4.00E+00 | 3.50E+00 | 0.54 | 0.52 | 17 |
1.00E+01 | 8.70E+00 | 0.94 | 0.91 | 21 |
5.00E+01 | 4.40E+01 | 1.64 | 1.69 | 21 |
1.00E+02 | 8.70E+01 | 1.94 | 2.04 | 21 |
1.00E+03 | 8.70E+02 | 2.94 | 3.01 | 21 |
1.00E+04 | 8.70E+03 | 3.94 | 3.9 | 21 |
1.00E+05 | 8.70E+04 | 4.94 | 4.88 | 21 |
1.00E+06 | 8.70E+05 | 5.94 | 5.87 | 21 |
1.00E+07 | 8.70E+06 | 6.94 | 6.92 | 21 |
1.00E+08 | 8.70E+07 | 7.94 | 8.01 | 21 |
1.00E+09 | 8.70E+08 | 8.94 | 9.04 | 21 |
2.00E+09 | 1.70E+09 | 9.24 | 9.38 | 21 |
TABLE 14 | ||||
Linearity in Serum | ||||
Assigned | Mean Log 10 | |||
Nominal Titer | Assigned Titer | Log10 | Titer | Repli- |
(IU/mL) | (IU/mL) | Titer | observed | cates |
4.00E+00 | 3.30E+00 | 0.52 | 0.7 | 21 |
1.00E+01 | 8.30E+00 | 0.92 | 0.99 | 21 |
5.00E+01 | 4.10E+01 | 1.62 | 1.73 | 21 |
1.00E+02 | 8.30E+01 | 1.92 | 2.03 | 21 |
1.00E+03 | 8.30E+02 | 2.92 | 2.93 | 21 |
1.00E+04 | 8.30E+03 | 3.92 | 3.8 | 21 |
1.00E+05 | 8.30E+04 | 4.92 | 4.78 | 21 |
1.00E+06 | 8.30E+05 | 5.92 | 5.75 | 21 |
1.00E+07 | 8.30E+06 | 6.92 | 6.73 | 21 |
1.00E+08 | 8.30E+07 | 7.92 | 7.78 | 21 |
1.00E+09 | 8.30E+08 | 8.92 | 8.92 | 21 |
2.00E+09 | 1.70E+09 | 9.22 | 9.22 | 21 |
Final Conc. in 50 | |
Reagent | ul-PCR (uM) |
Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) | 3′300 |
NaN3/Ri, buffered with 10 mM Tris at pH 7 | 0.018 |
pH: 6.41 |
Final Conc. in 50 | ||
Reagent | ul-PCR | |
Glycerol (%, w/v) | 3% | |
Tricine | 60 | mM |
DMSO (%, v/v) | 5.4% | |
KOAc | 120 | mM |
Tween 20 (v/v) | 0.015% | |
NTQ21-46 A | 0.222 | μM |
ZO5D | 0.9 U/μL (45 U/rxn) | |
UNG | 0.2 U/μL (10 U/rxn) | |
Sodium Azide (w/v) | 0.027 | |
dCTPs | 400 | μM |
dGTPs | 400 | μM |
dATPs | 400 | μM |
dUTPs | 800 | μM |
Primers/probes selected from SEQ ID NOs 60-76 | 0.1 | μM |
SEQ ID NO 42 | 0.3 | μM |
SEQ ID NO 43 | 0.3 | μM |
SEQ ID NO 44 | μM |
TABLE 15 | |||
Hit rates and Probit with 200 μL sample | |||
process input volume for EDTA-plasma | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
55 IU/mL | 21 | 21 | 100% |
38 IU/mL | 21 | 21 | 100% |
25 IU/mL | 21 | 20 | 95% |
12.5 IU/mL | 21 | 19 | 90% |
6 IU/mL | 21 | 15 | 71% |
3 IU/mL | 21 | 6 | 29% |
0 IU/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 17.4 IU/mL | ||
95% confidence interval for LOD by PROBIT | 12.1-34.3 | ||
analysis | IU/mL |
TABLE 16 | |||
Hit rates and Probit with 500 μL sample | |||
process input volume for EDTA-plasma | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
22 IU/mL | 21 | 21 | 100% |
15 IU/mL | 21 | 21 | 100% |
10 IU/mL | 20 | 20 | 100% |
5 IU/mL | 21 | 19 | 76% |
2.5 IU/mL | 21 | 15 | 71% |
1 IU/mL | 21 | 6 | 57% |
0 IU/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 9.0 IU/mL | ||
95% confidence interval for LOD by PROBIT | 5.5-25.4 | ||
analysis | IU/mL |
TABLE 17 | |||
Hit rates and Probit with 200 μL | |||
sample process input volume for serum | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
55 IU/mL | 21 | 21 | 100% |
38 IU/mL | 21 | 21 | 100% |
25 IU/mL | 21 | 20 | 95% |
12.5 IU/mL | 21 | 18 | 86% |
6 IU/mL | 21 | 13 | 62% |
3 IU/mL | 21 | 6 | 29% |
0 IU/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 20.2 IU/mL | ||
95% confidence interval for LOD by PROBIT | 14.0-39.3 | ||
analysis | IU/mL |
TABLE 18 | |||
Hit rates and Probit with 500 μL | |||
sample process input volume for serum | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
22 IU/mL | 21 | 21 | 100% |
15 IU/mL | 21 | 21 | 100% |
10 IU/mL | 21 | 20 | 95% |
5 IU/mL | 21 | 18 | 86% |
2.5 IU/mL | 21 | 12 | 57% |
1 IU/mL | 21 | 4 | 19% |
0 IU/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 8.2 IU/mL | ||
95% confidence interval for LOD by PROBIT | 5.8-15.0 | ||
analysis | IU/mL |
TABLE 19 | ||||
Linearity in EDTA Plasma | ||||
Assigned | Mean Log 10 | |||
Nominal Titer | Assigned Titer | Log10 | Titer | Repli- |
(IU/mL) | (IU/mL) | Titer | observed | cates |
8.00E+00 | 4.87E+00 | 0.7 | 0.6 | 15 |
1.00E+01 | 6.09E+00 | 0.8 | 0.8 | 17 |
1.00E+02 | 6.09E+01 | 1.8 | 1.7 | 21 |
1.00E+03 | 6.09E+02 | 2.8 | 2.8 | 21 |
1.00E+04 | 6.09E+03 | 3.8 | 3.8 | 21 |
1.00E+05 | 6.09E+04 | 4.8 | 4.7 | 21/20 |
1.00E+06 | 6.09E+05 | 5.8 | 5.6 | 21/20 |
1.00E+07 | 6.09E+06 | 6.8 | 6.7 | 21 |
1.00E+08 | 6.09E+07 | 7.8 | 7.8/7.7 | 21/18 |
2.00E+08 | 1.22E+08 | 8.1 | 8 | 21/20 |
TABLE 20 | ||||
Linearity in Serum | ||||
Assigned | Mean Log 10 | |||
Nominal Titer | Assigned Titer | Log10 | Titer | Repli- |
(IU/mL) | (IU/mL) | Titer | observed | cates |
8.00E+00 | 3.90E+00 | 0.6 | 0.7 | 10 |
1.00E+01 | 4.96E+00 | 0.7 | 0.7 | 14 |
1.00E+02 | 4.96E+01 | 1.7 | 1.6 | 21 |
1.00E+03 | 4.96E+02 | 2.7 | 2.8 | 21 |
1.00E+04 | 4.96E+03 | 3.7 | 3.7 | 21 |
1.00E+05 | 4.96E+04 | 4.7 | 4.7 | 21 |
1.00E+06 | 4.96E+05 | 5.7 | 5.7 | 21 |
1.00E+07 | 4.96E+06 | 6.7 | 6.7 | 21 |
1.00E+08 | 4.96E+07 | 7.7 | 7.7 | 21 |
2.00E+08 | 9.92E+07 | 8 | 8.1 | 21 |
Final Conc. in 50 | |
Reagent | ul-PCR (uM) |
Mn(Ac)2 * 4H2O (pH 6.1 adjusted with Acetic Acid) | 3′300 |
NaN3/Ri, buffered with 10 mM Tris at pH 7 | 0.018 |
pH: 6.41 |
Final Conc. in 50 | ||
Reagent | ul-PCR | |
Glycerol (%, w/v) | 3% | |
Tricine | 60 | mM |
DMSO (%, v/v) | 5.4% | |
KOAc | 120 | mM |
Tween 20 (v/v) | 0.02% | |
Aptamer NTQ21-46 A | 0.222 | μM |
ZO5D Polymerase | 0.9 U/μL (45 U/rxn) | |
UNG | 0.2 U/μL (10 U/rxn) | |
Sodium Azide (w/v) | 0.027 | |
dCTPs | 400 | μM |
dGTPs | 400 | μM |
dATPs | 400 | μM |
dUTPs | 800 | μM |
Primers/probes selected from SEQ ID NOs 1-35 | 0.1 μM-0.3 μM | |
SEQ ID NO 50 | 0.3 | μM |
SEQ ID NO 51 | 0.3 | μM |
SEQ ID NO 52 | μM |
TABLE 21 | |||
LOD analysis for 200 μL input volume in EDTA-plasma | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
200 cp/mL | 21 | 21 | 100% |
100 cp/mL | 21 | 21 | 100% |
80 cp/mL | 21 | 21 | 100% |
50 cp/mL | 21 | 20 | 95.2% |
30 cp/mL | 21 | 18 | 85.7% |
20 cp/mL | 21 | 17 | 81.0% |
10 cp/mL | 21 | 8 | 38.1% |
0 cp/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 41.8 cp/mL | ||
95% confidence interval for LOD by PROBIT | 30.9-74.9 | ||
analysis | cp/mL |
TABLE 22 | |||
LOD analysis for 500 μL input volume | |||
Number of | Number of | ||
Concentration | replicates | positives | Hit rate |
30 cp/mL | 21 | 21 | 100% |
25 cp/mL | 21 | 20 | 95.2% |
20 cp/mL | 21 | 21 | 100% |
13.5 cp/mL | 21 | 18 | 85.7% |
9 cp/mL | 21 | 13 | 61.9% |
6 cp/mL | 21 | 9 | 42.9% |
0 cp/mL (neg. control) | 21 | 0 | 0% |
LOD by PROBIT analysis (95% hitrate) | 18.9 cp/mL | ||
95% confidence interval for LOD by PROBIT | 14.9-29.4 | ||
analysis | cp/mL |
TABLE 23 | ||||
Linearity in EDTA Plasma | ||||
Assigned | Mean Log 10 | |||
Nominal Titer | Assigned Titer | Log10 | Titer | Repli- |
(cp/mL) | (cp/mL) | Titer | observed | cates |
1.50E+01 | 1.50E+01 | 1.2 | 1.3 | 21 |
2.00E+01 | 2.00E+01 | 1.3 | 1.5 | 21 |
5.00E+01 | 5.10E+01 | 1.7 | 1.8 | 21 |
1.00E+02 | 1.00E+02 | 2 | 2 | 21 |
1.00E+03 | 1.00E+03 | 3 | 3 | 21 |
1.00E+04 | 1.00E+04 | 4 | 4 | 21 |
1.00E+05 | 1.00E+05 | 5 | 5 | 21 |
1.00E+06 | 1.00E+06 | 6 | 6 | 21 |
1.00E+07 | 1.00E+07 | 7 | 7 | 21 |
2.00E+07 | 2.00E+07 | 7.3 | 7.4 | 21 |