Family of E6-2 amplification curves obtained by direct and nested qPCR. RFU, relative fluorescence units. Threshold (thick horizontal line): 50 RFU. (A) Family of curves obtained in direct qPCR mixtures with pHV101 dilutions and primers for E6-2. Curve 1, 2.5 × 10⁶ pHV101 copies. Curve 2, 2.5 × 10⁵ copies. Curve 3, 2.5 × 10⁴ copies. Curve 4, 2.5 × 10³ copies. Curve 5, Blank (without DNA). (B) Family of curves obtained by nested qPCR with E6-2 primers and 1/50 volume of mixtures preamplified 15 cycles with pHV101 and E6-1 primers. Curve 1, 2.5 × 10⁶ pHV101 copies. Curve 2, 2.5 × 10⁵ copies. Curve 3, 2.5 × 10⁴ copies. Curve 4, 2.5 × 10³ copies. Curve 5, 2.5 × 10² copies. Curve 6, blank (without DNA).

Thermal denaturation (fluorescence extinction) profiles of the E6-2 amplicons generated from pHV101 and SiHa DNA. RFU, relative fluorescence units. (A) Family of curves obtained with serially diluted pHV101 or 50 ng of SiHa DNA. Threshold (thick horizontal line): 50 RFU. Curve 1, 2.5 × 10⁶ pHV101 copies. Curve 2, 2.5 × 10⁵ pHV101 copies. Curve 3, 2.5 × 10⁴ pHV101 copies. Curve 4, 2.5 × 10³ pHV101 copies. Curve 5, 2.5 × 10²pHV101 copies. Curve 6, Blank (without DNA). Curve 7, normal human blood DNA. Curve 8, 50 ng of SiHa cell DNA with the pU1M/pU1R primer pair. Curve 9, 50 ng of SiHa cell DNA with the pU1M forward primer only. (B) Thermal fluorescence extinction profiles of nested qPCR products. Profiles of pHV101 and SiHa DNA mixtures with both primers (peaks at 85.5°C), mixtures without DNA or with normal human blood DNA with both primers (peaks at 78.5°C), and SiHa DNA with the pU1M primer only (nearly horizontal line close to the baseline).

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.

Sequences of the E6-1 and E6-2 amplicons generated from pHV101. (A) Diagram of the HPV16 long control region (LCR) and the E6 and E7 genes, whose sequences overlap partially. The E6-1 amplicon (645 bp) is generated first by direct PCR with the LCRS/E7AS primer pair and then the E6-2 (237 bp) amplicon is generated by nested PCR with the pU1M/pU2R primer pair. The scale in the lower part of the figure indicates the positions of LCR, the E6 and E7 oncogenes and the E6-1 and E6-2 amplicon sequences on the HPV16 genome. (B) The E6-645 insert sequence corresponds to the cloned E6-1 amplicon, spans nucleotides 26-671 (645 bp) of the HPV16 genome and is flanked by the LCRS and E7AS primers (white letters on black background) at the 5' and 3' ends, respectively; it contains 38% GC and is 99% identical to the E6-HPV16 ORF [10]. The fragment corresponding to the E2-237 amplicon (in italics) spans nucleotides 419-656 (237 bp) of the HPV16 genome and is flanked by the pU1M and pU2R primers (white italics on black background), respectively; its sequence contains 41% GC and is identical to the corresponding ORF sequence of the E6-HPV16 oncogene. The three gray italic letters on black background correspond to the overlapping bases of the pU2R and E7AS primers.