Table 1.
RT-ddPCR reaction conditions and quantification results
| Figure | Procedure | RT enzyme | Priming method | PCR enzyme | SIV input (copies) | SIV count (copies) | PCR thermal cycling condition |
|---|---|---|---|---|---|---|---|
| 1A | One step | SSIII | Gene-specific | Platinum Taq | 0 | N.D | 50 °C 30 min, 94 °C 7 min, 38× (94 °C 14 s, 60 °C 30 s, 68 °C 30 s, 94 °C 1 s), 94 °C 14 s, 60 °C 30 s, 68 °C 5 min 30 s, 98 °C 10 min, 4 °C hold |
| 1B | 10 | N.D | |||||
| 1C | 0 | N.D | |||||
| 1D | 10 | N.D | |||||
| 1E | 0 | N.D | |||||
| 1F | 10 | N.D | |||||
| 1G | 0 | N.D | |||||
| 1H | 10 | N.D | |||||
| 2A | Two step | M-MLV | Gene-specific | AmpliTaq Gold | 0 | 0 | (RT) 25 °C 15 min; 37 °C 60 min; 90 °C 30 min; 25 °C 30 min; 4 °C hold. (PCR) 95 °C 7 min, 40× (95 °C 15 s, 60 °C 1 min), 98 °C 10 min, 4 °C hold |
| 2B | 10 | 14 | |||||
| 2C | SSIII | 0 | 1 | (RT) 25 °C 15 min; 37 °C 60 min; 85 °C 5 min; 25 °C 30 min; 4 °C hold. (PCR) 95 °C 7 min, 40× (95 °C 15 s, 60 °C 1 min), 98 °C 10 min, 4 °C hold | |||
| 2D | 10 | 14 | |||||
| 2E | M-MLV | Random hexamer | 0 | 0 | (RT) 25 °C 15 min; 50 °C 50 min; 90 °C 30 min; 25 °C 30 min; 4 °C hold. (PCR) 95 °C 7 min, 40× (95 °C 15 s, 60 °C 1 min), 98 °C 10 min, 4 °C hold | ||
| 2F | 10 | 1 | |||||
| 2G | SSIII | 0 | 1 | (RT) 25 °C 15 min; 50 °C 50 min; 85 °C 5 min; 25 °C 30 min; 4 °C hold. (PCR) 95 °C 7 min, 40× (95 °C 15 s, 60 °C 1 min), 98 °C 10 min, 4 °C hold | |||
| 2H | 10 | 2 | |||||
| 3A | Two-step | SSIII | Gene-specific | AmpliTaq Gold | 0 | N.D | (RT) 25 °C 15 min; 50 °C 50 min; 85 °C 5 min; 25 °C 30 min; 4 °C hold. (PCR) 95 °C 7 min, 40× (95 °C 15 s, 60 °C 1 min), 98 °C 10 min, 4 °C hold |
| 3B | 100 | N.D | |||||
| 3C | Random hexamer | 0 | N.D | ||||
| 3D | 100 | N.D | |||||
| 4A | Two-step | SSIV | Gene-specific | AmpliTaq Gold | 0 | 0 | (RT) 25 °C 15 min; 50 °C 10 min; 95 °C 10 min; 25 °C 30 min; 4 °C hold. (PCR) 95 °C 7 min, 40× (95 °C 15 s, 60 °C 1 min), 98 °C 10 min, 4 °C hold |
| 4B | 10 | 10 |
Reaction conditions (including the specific procedure applied (i.e. one-step vs. two-step RT-ddPCR), the reverse transcriptase (RT) enzyme used at the RT step, the priming method, the enzyme used at the ddPCR step, and the PCR thermal cycling condition) and quantification results (including SIV input (copies) and count (copies)) are listed. N.D., quantitation not done due to background signals