Quantification of Streptococcus salivarius using the digital polymerase chain reaction as a liver fibrosis marker

Figure 1 Specificity of primers and probes for Streptococcus salivarius. Positive droplets of Streptococcus salivarius with channel amplitude signals (Blue signal: tnpA; Green signal: 16S rRNA). A: Digital PCR (dPCR) assay targeting both the tnpA and 16S rRNA gene to 8 kinds of Streptococcus spp.; B: dPCR assay targeting both the tnpA and 16S rRNA gene to 8 kinds of common microorganisms of the genitourinary tract.

Figure 2 Comparison of bacterial load measured using digital PCR and bacterial titremeasured using plaque assay. A: Profiles of digital PCR (dPCR) assays targeting the tnpA on bacterial suspensions of Streptococcus salivarius (S. salivarius) strain; B: Profiles of dPCR assays targeting 16S rRNA in S. salivarius bacterial suspensions. Fluorescence amplitude plot of dPCR assays on bacterial suspensions at 5-fold diluted concentrations from 2.00 × 10⁶ to 1.28 × 10² colony forming unit/mL. The blue line represents a threshold to distinguish between positive and negative droplets based on the fluorescence signal. Droplets with a fluorescence signal above this threshold are classified as positive, indicating the presence of amplification. dPCR: Digital PCR; CV: Coefficients of variability; CFU: Colony forming unit.

Figure 3 Comparison of bacterial load measured using digital PCR and quantitative PCR. A: Quantitative PCR (qPCR) can reliably detect bacterial concentrations down to 6.40 × 10² colony forming unit (CFU)/mL using a 1 μL sample volume; B: Digital PCR has a higher sensitivity than qPCR, as it can detect lower concentrations of bacteria (6.40 × 10² CFU/mL) with greater accuracy and precision. dPCR: Digital PCR; qPCR: Quantitative PCR; CFU: Colony forming unit.

Figure 4 Correlation between the bacterial load. A: Correlation between fibrosis stage and the bacterial load measured using digital PCR (dPCR) with tnpA; B: Correlation between fibrosis stage and the bacterial load measured using dPCR with 16S rRNA; C: Comparisons between mild and severe fibrosis in the bacterial load measured using dPCR with tnpA; D: Comparisons between mild and severe fibrosis in the bacterial load measured using dPCR with 16S rRNA.

Figure 5 Severe fibrosis receiver operating characteristic curves for patients with chronic liver disease. AUC: Area under the curve.