Fig. 3: Parameters optimization of MPHAC.

a–c Simulated results of amplification efficiency (a), specificity (b) and odds ratio (OR) (c) under different primer free energies based on the theoretical model. Details of the model construction are provided in Supplementary Section 3. The white dashed box represents the free energy range for 80% of FL-primers in 100,000-plex primer pairs. Amplification efficiency positively correlates with free energy, while specificity exhibits the opposite trend. We considered the amplification efficiency and specificity together by OR, and the results suggest that the primer free energy can be optimized around −11.5 kcal mol⁻¹. (d). e OR values under different length FL-primers and FE-primers (d) and different primer free energies (e). Here, the coefficient of variation (CV) is a measure of the relative dispersion of a data set, expressed as the ratio of the standard deviation to the mean. f Primer length and free energy distribution. Six sets of FE-primers and six sets of FL-primers were designed based on plasmid sequences. g Monitoring primer hybridization fluorescence kinetics using X-probe. Experimental data are represented by dots, and simulated data is depicted by lines. h Simulated hybridization yields for primers when A = B (left) and A > > B (right). Here, A refers to the primer, and B denotes the reverse complementary sequence of the primer (template). The range of hybridization yields for FE-primers is considerably narrower (pink) than that of FL-primers (blue). i PCR results for FE- and FL-primers. Data are presented as mean ± standard deviation (SD), based on measurements from 3 independent samples. Here, Ct value (Cycle threshold value) represents the cycle number at which the fluorescence signal exceeds a defined threshold, indicating the presence of the target. Theoretically, identical concentrations of the target should result in the same Ct value. Source data are provided as a Source Data file.