Thermomechanical DNA extraction from infected dental pulp for next-generation sequencing applications

DNA extraction from infected dental pulp tissue represents a critical methodological limitation in molecular endodontics, severely constraining pathogen identification and precision therapeutic approaches. Conventional extraction protocols demonstrate systematic failures when applied to inflamed pulp samples containing complex hydroxyapatite-collagen matrices, neutrophil extracellular traps, and inflammatory mediators that compromise nucleic acid integrity and downstream next-generation sequencing applications.

The present investigation comprehensively validated a thermomechanical extraction protocol combining optimized extended thermal incubation with intensive mechanical disruption cycles specifically designed for infected dental pulp tissues. Performance was systematically evaluated against the current standardised systems using multi-parameter quality assessment, statistical analysis including effect size calculations across 24 infected pulp samples from patients with irreversible pulpitis.

The thermomechanical protocol achieved transformative improvements across all performance metrics: 3.7-fold enhancement in DNA concentration (69.8 ± 10.21 vs. 18.83 ± 12.72 ng/μL, p < 0.01, Cohen's d = 4.2), 18 % improvement in protein purity ratios (A₂₆₀/A₂₈₀: 2.23 ± 0.23 vs. 1.89 ± 0.060, p < 0.01), and unprecedented 4-6 fold enhancement in inter-sample reproducibility (coefficient of variation reduction from 67.6 % to 14.6 %). Universal optimal quality classification was achieved (100 % vs. 58.3 % for conventional protocols), enabling reliable endodontic microbiome analysis and pulp genomics applications.

The current thermomechanical approach establishes performance standards for infected dental pulp DNA extraction, providing reliable methodology for endodontic microbiome analysis, pulp-dentin genomics, and precision therapy selection. The superior reproducibility and pathophysiology-specific refinement positions it as essential for advancing molecular endodontics and evidence-based therapeutic decision-making.