Development of a qPCR-based method for absolute quantification of facial skin bacteria considering DNA extraction...

The skin microbiome differs with age, and understanding the relationship between the microbial balance and skin health is valuable in skin healthcare. In this study, we developed a quantitative real-time PCR (qPCR) method using TaqMan MGB probes to measure the abundance of indigenous skin bacteria. Representative facial skin bacteria (Cutibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus) were detected and quantified with high sensitivity using specific primer sets and probes. C. acnes was divided into two groups for qPCR analysis: Group 1, which included major acne-associated types, and Group 2, which included C. acnes subsp. defendens, which is predominant in healthy skin. Cell suspensions of each bacterial species, for which the total cell numbers were assessed, were subjected to DNA extraction with lytic enzyme treatment. The purified DNA enabled the accurate calculation of the extraction efficiency. The DNA extraction efficiency of the targeted skin bacteria ranged from 30% to 100%, indicating a species-dependent variation. The established method enabled stable quantification over a bacterial concentration range of 10⁴-10⁸ cells/mL. The qPCR results using DNA extracted from mixed bacterial suspensions were generally consistent with the expected genomic DNA copy numbers after correcting for species-specific DNA extraction efficiencies. The established extraction efficiency-corrected qPCR method provides improved quantification of bacterial abundance on facial skin.