Comparison of Reduced PCR Volume PowerPlex Fusion 6C Kit Validations on Manual and Automated Systems
Abstract
:1. Introduction
2. Materials and Methods
2.1. Samples
2.2. DNA Extraction
2.3. PCR Amplification and Settings
2.4. Capillary Electrophoresis
3. Validation
3.1. Repeatability and Reproducibility
3.2. Sensitivity
3.3. Mixture Samples
3.4. Stability
3.5. Degraded Casework Samples
4. Results
4.1. Sensitivity
4.2. Mixture Analysis
4.3. Stability Assessment
4.4. Degraded Casework Samples
5. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Input DNA (ng) | Manual (M) Automated (A) | Blue Dye | Green Dye | Black Dye | Red Dye | Purple Dye | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
1 | M (mean ± SD. RFU) | 13,796.3 | 2639.2 | 9732.1 | 1829.2 | 12,012.2 | 2275.3 | 12,571.4 | 3246.8 | 7838 | 820.6 |
M (DO%) | 0 | 0 | 0 | 0 | 0 | ||||||
0.5 | M (RFU) | 8221.8 | 2382.1 | 6062.4 | 1511.3 | 5900.6 | 1288.9 | 6291.2 | 1534.4 | 4385.1 | 803.9 |
M (DO%) | 0 | 0 | 0 | 0 | 0 | ||||||
A (mean ± SD. RFU) | 6099.3 | 3001.1 | 4420.9 | 2606.7 | 5262.7 | 2191.6 | 5897 | 2566 | 3112.1 | 2236.2 | |
A (DO%) | 0 | 0 | 0 | 0 | 0 | ||||||
0.15 | M (mean ± SD. RFU) | 2583.5 | 1224.9 | 1959.7 | 1279.6 | 1592 | 605.7 | 1813.5 | 712.3 | 1162.7 | 964.3 |
M (DO%) | 0 | 0 | 0 | 0 | 0 | ||||||
A (mean ± SD. RFU) | 1808.2 | 1131.8 | 1070.2 | 755.4 | 1546.2 | 867.9 | 1687.6 | 848 | 530.1 | 386.7 | |
A (DO%) | 0 | 0 | 0 | 0 | 10 | ||||||
0.075 | M (mean ± SD. RFU) | 1119.2 | 652.7 | 993.7 | 682.7 | 761.6 | 384 | 729.3 | 307.3 | 583.2 | 381.7 |
M (DO%) | 0 | 4 | 5 | 8 | 10 | ||||||
0.0375 | M (mean ± SD. RFU) | 478.6 | 322.6 | 425.1 | 355.9 | 407.2 | 293.1 | 365.1 | 275.2 | 264.8 | 173.2 |
M (DO%) | 13 | 4 | 5 | 6 | 10 | ||||||
0.015 | M (mean ± SD. RFU) | 127 | 46.6 | 109.6 | 53.2 | 111.6 | 63 | 112.1 | 42.9 | 58.5 | 0.7 |
M (DO%) | 68 | 70 | 60 | 66 | 80 | ||||||
0.0075 | M (mean ± SD. RFU) | 114.6 | 83.1 | 103.9 | 41.2 | 98.1 | 34.9 | 136.2 | 43.7 | 79.7 | 13.1 |
M (DO%) | 82 | 76 | 73 | 90 | 70 |
Input DNA (ng) | Manual (M) Automated (A) | Blue Dye | Green Dye | Black Dye | Red Dye | Purple Dye | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
0.15 | M (mean ± SD. RFU) | 7721.6 | 2223.5 | 5917.6 | 1952.5 | 6628.5 | 2038.6 | 6480.7 | 1874.9 | 4217.7 | 1344.9 |
M (DO%) | 0 | 0 | 0 | 0 | 0 | ||||||
A (mean ± SD. RFU) | 9138.8 | 3302.2 | 6454.7 | 3035.6 | 7498.6 | 2395.3 | 7648.6 | 2486.3 | 5300.6 | 1945.3 | |
A(DO%) | 0 | 0 | 0 | 0 | 0 | ||||||
0.075 | M (mean ± SD. RFU) | 4846.1 | 2255 | 3672.9 | 1476.5 | 3652.8 | 1179.3 | 4067.2 | 1652.2 | 2474.6 | 1056.4 |
M (DO%) | 2 | 0 | 0 | 0 | 0 | ||||||
A (mean ± SD. RFU) | 4293.3 | 1990.4 | 2936.3 | 1612.7 | 3555.8 | 1511.2 | 3799 | 1847.7 | 2082.6 | 642.3 | |
A (DO%) | 0 | 0 | 2 | 0 | 0 | ||||||
0.0375 | M (mean ± SD. RFU) | 2291.9 | 1108.4 | 1637.2 | 747.2 | 1723.7 | 979.9 | 1734.8 | 1033.1 | 853.6 | 673.1 |
M (DO%) | 2 | 2 | 0 | 0 | 0 | ||||||
A (mean ± SD. RFU) | 1520.7 | 842.1 | 1025.9 | 598.8 | 1255.6 | 671.2 | 1423.8 | 805.5 | 749.4 | 384.7 | |
A (DO%) | 7 | 4 | 7 | 4 | 0 | ||||||
0.015 | M (mean ± SD. RFU) | 720.3 | 502.1 | 480.2 | 298.3 | 653.4 | 373.7 | 671.6 | 358.7 | 388.3 | 219.4 |
M (DO%) | 15 | 14 | 18 | 18 | 10 | ||||||
A (mean ± SD. RFU) | 431 | 542.2 | 474.3 | 360.8 | 508.1 | 377 | 650.5 | 515.1 | 368 | 218.3 | |
A(DO%) | 43 | 40 | 35 | 46 | 40 | ||||||
0.0075 | M (mean ± SD. RFU) | 385.5 | 375.7 | 284.9 | 216.8 | 348.8 | 241 | 396.4 | 328.8 | 325 | 165.4 |
M (DO%) | 38 | 48 | 43 | 36 | 40 | ||||||
M (mean ± SD. RFU) | 294.1 | 176.2 | 273.5 | 186.5 | 326.7 | 291 | 403.7 | 225.2 | 329 | NA | |
M(DO%) | 53 | 72 | 55 | 38 | 90 |
Manually Prepared PCR | Automated PCR | |||
---|---|---|---|---|
Mix (Female:Male) | Allele Dropout (No.) | Allele Dropout in % | Allele Dropout (No.) | Allele Dropout in % |
1:1 | 0 | 0.0% | 0 | 0.0% |
1:4 | 0 | 0.0% | 2 | 0.89% |
1:7 | 1 | 0.44% | 7 | 3.11% |
1:9 | 5 | 2.22% | 18 | 8.00% |
1:19 | 32 | 14.22% | 52 | 23.11% |
DI | DNA Concentration (ng/µL) | Average DNA Concentration (ng/µL) | Samples (No.) | DNA Profile Suitable for Comparison | Average Locus Dropout (%) | ||
---|---|---|---|---|---|---|---|
Manual | Hamilton Biorobot | Total | |||||
LOW (3–4) | 0.002–0.1465 | 0.0222 | 92 | 60.98% | 50% | 59.78% | 52.22% |
MEDIUM (4–7) | 0.0031–0.1423 | 0.0175 | 80 | 46.67% | 40% | 46.25% | 64.24% |
HIGH (>7) | 0.0021–0.1418 | 0.0151 | 29 | 26.92% | NA | 24.14% | 83.65% |
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Lőrincz, E.É.; Mátrai, N.; Rádóczy, K.A.; Cseppentő, T.; Magonyi, N.M.; Heinrich, A. Comparison of Reduced PCR Volume PowerPlex Fusion 6C Kit Validations on Manual and Automated Systems. DNA 2024, 4, 52-63. https://doi.org/10.3390/dna4010003
Lőrincz EÉ, Mátrai N, Rádóczy KA, Cseppentő T, Magonyi NM, Heinrich A. Comparison of Reduced PCR Volume PowerPlex Fusion 6C Kit Validations on Manual and Automated Systems. DNA. 2024; 4(1):52-63. https://doi.org/10.3390/dna4010003
Chicago/Turabian StyleLőrincz, Eszter É., Norbert Mátrai, Katalin A. Rádóczy, Tamás Cseppentő, Nóra M. Magonyi, and Attila Heinrich. 2024. "Comparison of Reduced PCR Volume PowerPlex Fusion 6C Kit Validations on Manual and Automated Systems" DNA 4, no. 1: 52-63. https://doi.org/10.3390/dna4010003