Figure 4.

Complete and minimum E6-1 preamplification mixtures used to perform E6-2 nested qPCR amplification. (A) Complete preamplification series. Successive stages: 1) preparation of E6-1 preamplification mixture containing; 2) "preamplification" by conventional PCR; 3) E6-2 amplification in "nested" qPCR mixtures containing EvaGreen and 1/50 volume of the E6-1 preamplified mixture. Tubes 1, a, b, c and d: positive control preamplification mixtures with serial logarithmic dilutions of pHV101 in the range of 2.5 × 10⁶-2.5 × 10² molecules per tube. Tube 2: Blank preamplification (without DNA). Tube 3: Problem preamplification (50 ng of SiHa DNA). Tubes 4 and 5: Negative preamplification controls (50 ng "carrier" normal human blood DNA). Asterisks indicate preamplified mixtures. (B) Minimum preamplification series. Successive stages: 1) preparation of E6-1 preamplification mixture including only the positive control ("calibration") with the highest pHV101 content; 2a) E6-1 "preamplification" by conventional PCR; 2b) serial logarithmic dilutions of the preamplified calibration mixture; 3) amplification of E6-2 in nested qPCR mixtures containing EvaGreen, the E6-2 primers and 1/50 volume of E6-1 preamplified mixtures. Tube 1: Positive control amplification mixture with 2.5 × 10⁶ pHV101 molecules. Tube 2: Blank preamplification mixture (without DNA). Tube 3: Problem preamplification mixture (50 ng of SiHa DNA). Tubes 4 and 5: Negative preamplification controls (50 ng "carrier" normal human blood DNA). Tubes a, b, c, and d: serial logarithmic dilutions from the preamplified positive control mixture used to prepare nested qPCR mixtures equivalent to those preamplified with 2.5 × 10⁵-2.5 × 10² pHV101 molecules. Asterisks of numbered tubes indicate preamplified mixtures.