Characteristics of HLA- and Y-specific primer sets and PCR assays
| PCR target . | Amplicon, base pairs . | Concentration, pmol/μL . | Sensitivity,*†‡ copies/% . | Specificity,‡§ Ct value pos/neg . | PCR efficiency, % . |
|---|---|---|---|---|---|
| HLA-A*01 | 205 | 30 | 1.7 ± 1.0 (0.006% ± 0.004%) | 20.7 / 39.1 (Δ = 18.5) | 117.2 ± 15.3 |
| HLA-A*02 | 205 | 10 | 1.7 ± 1.0 (0.006% ± 0.004%) | 21.7 / 40.0 (Δ = 18.3) | 107.4 ± 12.0 |
| HLA-A*24 | 125 | 30 | 2.0 ± 2.2 (0.007% ± 0.008%) | 22.9 / 37.4 (Δ = 14.5) | 127.1 ± 9.0 |
| HLA-B*51/52 | 120 | 10 | 2.2 ± 0.9 (0.008% ± 0.003%) | 22.6 / 36.4 (Δ = 13.8) | 124.9 ± 15.0 |
| HLA-B*08 | 105 | 20 | 1.7 ± 1.0 (0.006% ± 0.004%) | 21.6 / 38.2 (Δ = 16.6) | 120.3 ± 8.8 |
| HLA-DRB1*04 | 100 | 50 | 4.0 ± 1.6 (0.014% ± 0.006%) | 23.8 / 40.0 (Δ = 16.2) | 131.2 ± 12.0 |
| HLA-DRB1*10 | 90 | 20 | 1.3 ± 0.9 (0.005% ± 0.003%) | 20.0 / 37.4 (Δ = 17.3) | 131.0 ± 6.2 |
| HLA-DRB1*13 | 130 | 20 | 2.0 ± 2.2 (0.007% ± 0.008%) | 22.8 / 38.5 (Δ = 15.7) | 145.4 ± 3.9 |
| HLA-DRB1*16 | 145 | 20 | 1.7 ± 1.0 (0.006% ± 0.004%) | 20.3 / 34.2 (Δ = 13.9) | 116.4 ± 5.4 |
| DYZ1 | 86 | 20 | ≤ 1.0 ± 0.0 (≤ 0.004% ± 0.0%) | 19.4 / 33.4 (Δ = 14.0) | 117.9 ± 6.0 |
| HCK | 81 | 20 | 20.0 / – (–) |
| PCR target . | Amplicon, base pairs . | Concentration, pmol/μL . | Sensitivity,*†‡ copies/% . | Specificity,‡§ Ct value pos/neg . | PCR efficiency, % . |
|---|---|---|---|---|---|
| HLA-A*01 | 205 | 30 | 1.7 ± 1.0 (0.006% ± 0.004%) | 20.7 / 39.1 (Δ = 18.5) | 117.2 ± 15.3 |
| HLA-A*02 | 205 | 10 | 1.7 ± 1.0 (0.006% ± 0.004%) | 21.7 / 40.0 (Δ = 18.3) | 107.4 ± 12.0 |
| HLA-A*24 | 125 | 30 | 2.0 ± 2.2 (0.007% ± 0.008%) | 22.9 / 37.4 (Δ = 14.5) | 127.1 ± 9.0 |
| HLA-B*51/52 | 120 | 10 | 2.2 ± 0.9 (0.008% ± 0.003%) | 22.6 / 36.4 (Δ = 13.8) | 124.9 ± 15.0 |
| HLA-B*08 | 105 | 20 | 1.7 ± 1.0 (0.006% ± 0.004%) | 21.6 / 38.2 (Δ = 16.6) | 120.3 ± 8.8 |
| HLA-DRB1*04 | 100 | 50 | 4.0 ± 1.6 (0.014% ± 0.006%) | 23.8 / 40.0 (Δ = 16.2) | 131.2 ± 12.0 |
| HLA-DRB1*10 | 90 | 20 | 1.3 ± 0.9 (0.005% ± 0.003%) | 20.0 / 37.4 (Δ = 17.3) | 131.0 ± 6.2 |
| HLA-DRB1*13 | 130 | 20 | 2.0 ± 2.2 (0.007% ± 0.008%) | 22.8 / 38.5 (Δ = 15.7) | 145.4 ± 3.9 |
| HLA-DRB1*16 | 145 | 20 | 1.7 ± 1.0 (0.006% ± 0.004%) | 20.3 / 34.2 (Δ = 13.9) | 116.4 ± 5.4 |
| DYZ1 | 86 | 20 | ≤ 1.0 ± 0.0 (≤ 0.004% ± 0.0%) | 19.4 / 33.4 (Δ = 14.0) | 117.9 ± 6.0 |
| HCK | 81 | 20 | 20.0 / – (–) |
neg indicates negative; and pos, positive.
Sensitivity was determined by diluting positive DNA in 2 different negative, background DNA samples, each in duplicate. Column represents geometric means ± SD.
One copy represents 7.1 pg of DNA; 0.01% means detection of 1 copy in a background of 10 000; 0.005% means 1 in 20 000.
A threshold of 75.0 was used in the qPCR software.
For each primer set 0.2 μg of 2 different positive DNA samples and 4 different negative DNA samples was amplified. Column represents geometric means ± SD.