Development and optimization of a simian immunodeficiency virus (SIV) droplet digital PCR (ddPCR) assay
Fig 1
Performance of SIV single quencher probe assay on Raindance ddPCR.
(A-D) Final SIV probe concentration varied from 50nM (A) to 200nM (D). (E) AptaTaq amount in this reaction was increased to 2U (compared to A-D and F, 1U AptaTaq in each reaction). (F) Final MgCl2 concentration was increased to 5.5mM (compared to A-E, 4.5mM MgCl2 in each reaction). (G) Single quencher probe SIV assay in TaqMan Universal mastermix. (H) Single quencher probe SIV assay in Quantabio Toughmix. For reactions performed in qPCR mastermix (A-F), MgCl2 concentration, primer, probe concentrations (in nM) and enzyme amount are indicated for each reaction in the corresponding plot’s upper right corner. Primer and probe concentrations were in the following order using 1A as an example: SIV (assay) 600 (nM, forward primer) 600 (nM, reverse primer) 50 (nM, probe). For reactions involving commercial mastermixes, only primer and probe concentrations are indicated. SIV DNA standard input in each reaction was 10000 copies. Additional reaction condition information (including template input and thermal cycling condition) is listed in Table 1.