Table 2.
Hepatitis E virus quantification by RT-dPCR on standard materials used for RT-qPCR assays.
| HEV genome copies/μl (OD) | RT-qPCR HEV genome copies/assay (5 μl) | RT-dPCR Theoretical expected quantification (OD) HEV genome copies/assay (0.38 μl) | RT-dPCR Absolute quantification HEV genome copies/assay | % of quantification of expected input standard by RT-dPCR |
|---|---|---|---|---|
| 250 | 1250 (3/3)0 | 95 | 35.5 ± 13.1 (3/3) | 37.4 |
| 125 | 625 (3/3) | 48 | 12.0 ± 6.3 (3/3) | 25.0 |
| 25 | 125 (3/3) | 10 | 3.0 ± 1.4 (3/3) | 30.0 |
| 13 | 065 (3/3) | 5 | 1.0 ± 1.4 (3/3) | 20.0 |
| 3 | 015 (2/3) | 1 | 0.3 ± 0.5 (1/3) | 30.0 |
The number of HEV genome copies (GC) of standard materials was determined by OD (spectrophotometer). The number of HEV GC per RT-qPCR assay and the theoretical expected HEV GC per RT-dPCR were calculated from OD by taking into account the volume of each assay (5 μl per RT-qPCR assay vs. 0.38 μl RT-dPCR per assay). The absolute quantification of HEV obtained by RT-dPCR was then compared to the theoretical expected HEV GC obtained by OD. The percentage of HEV quantification by RT-dPCR was calculated as follows: absolute quantification (RT-dPCR)/theoretical expected quantification (OD) X 100. All experiments were performed three times in duplicate. The number of positive assays is given in brackets and is shown in bold for the results corresponding to the limit of detection of the assay.