FIGURE 3.

Sensitivity of each primer/probe sets along with that of E6/E7 primer/probe sets against the 31 clinically known HPV-positive head and neck squamous cell carcinoma samples among DNA derived from healthy individual. A, Sensitivity of samples against each planned primer/probe sets along with E6/E7 primer/probe sets. To calculate the threshold, we prepared 32 clinically known HPV-positive head and neck squamous cell carcinoma anonymized samples that were serially diluted to 150, 30, 15, 6, 1.2, 0.6, and 0.3 cells (corresponding to 990 pg., 198 pg, 99 pg, 39.6 pg, 7.9 pg, 3.2 pg, and 1.6 pg of tumor weight, respectively) among 10-ng normal human DNA. Each qPCR was studied thrice, and the lowest dilution series showing three consistent reactions was considered as the threshold. Sensitivity was calculated as follows: (a) white, 100 cells (corresponds to 660 pg of tumor weight), (b) gray, 10 cells (corresponds to 66 pg of tumor weight), and (c) black, 1 cell (corresponds to 6.6 pg of tumor weight). The “BR E1-5” primer/probe sets showed the highest sensitivity (28.1%) for one cell among these eight primer/probe sets. B, The sensitivity of the combination of “BR E1-5” and “HR E5L2-10” primer/probe sets was superior to that of E6 & E7 combination at all concentrations (100 cells, 30 cells, 10 cells, 3 cells, 1 cell). Experiments were performed in triplicates