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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