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Table 1 RT-ddPCR reaction conditions and quantification results

From: Development of a reverse transcription droplet digital PCR (RT-ddPCR) assay for sensitive detection of simian immunodeficiency virus (SIV)

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
  1. 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
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