Table 1.
Sequences of primers used in this study.
Table 2.
Primer-probe sets used in the present study.
Fig 1.
Evaluation of the amplification efficacy of primer-probe sets by droplet digital PCR (ddPCR).
Absolute positive droplet counts (APD) and mean fluorescent intensity (MFI) were assessed by droplet digital PCR (ddPCR), which were compared among 20 prime-probe sets targeting the same E. histolytica gene (X64142). Assays were performed using different PCR cycles: 50 cycles (A) and 30 cycles (B). All conditions other than PCR cycles were the same for all experiments. MFIs were compared by one-way ANOVA and presented as compact letter display (CPD) using GraphPad Prism software.
Fig 2.
Evaluation of the amplification efficacy of five selected primer-probe sets at different annealing temperatures: 59°C versus 62°C.
Fig 3.
Evaluation of the diagnostic efficacy of TaqMan-probed quantitative PCR (qPCR).
Ct values of five primer-probe sets were compared by the serial dilution of E. histolytica DNA. PCR was performed with 40 cycles at different annealing temperatures: 59°C (A), or 62°C (B).
Fig 4.
Primer-probe spefific Ct cut-off values for the laboratory strain of E. histolytica (A) and evaluation using clinical samples (B).
A standard curve with 95% confidence intervals was drawn between Ct value by qPCR and square APD by ddPCR using a serial dilution of E. histolytica laboratory strain (R² = 0.9965, p < 0.0001). The primer-probe set 5 specific cut-off Ct value was determined as 36 cycles, which is the intersection of the standard curve on 1 APD by ddPCR (A). The APD and Ct value of clinical samples were directly plotted on the standard curve created in Fig 4A. Open triangles represent the results of the samples with a lower Ct than the cut-off Ct value (Ct ≤ 36 by qPCR). Filled triangles represent the results of the samples with a higher Ct than the cut-off Ct value (Ct > 36, or undetermined by qPCR). The samples plotted far outside the 95% CI are indicated by # (discordant PCR results), some of which were transferred to the shotgun metagenome analysis to check the existence of Entamoeba derived DNA (B).
Table 3.
Read sequence analysis of the template solutions.