Development of real‐time <scp>PCR</scp> and droplet digital <scp>PCR</scp> assays for the simultaneous quantification of bacterial and human mitochondrial <scp>DNA</scp> for forensic analysis

Forensic evidence recovered from crime scenes often contains a mixture of human and bacterial DNA. Although short tandem repeat (STR) profiling of genomic DNA (gDNA) is widely used for human identification, its effectiveness can be limited in cases involving highly degraded DNA. In such cases, human mitochondrial DNA (mtDNA) and microbiome analysis may serve as alternative methods. In this study, we developed a multiplex quantification assay targeting the bacterial 16S rRNA V7 region and the human mitochondrial NADH-dehydrogenase subunit 5 (ND5) gene. Quantification was performed using TaqMan-based real-time PCR (Human-Bacteria qPCR; HBQ) and droplet digital PCR (Human-Bacteria ddPCR; HBD). Optimal primer and probe concentrations were at 7 μM for the HBQ assay, and 5 μM bacterial primer set, 7 μM human mtDNA primer set, and 700 nM probes for the HBD assay. Sensitivity testing showed that the HBQ assay detected all DNA samples-except G147A-down to 20 fg, while the HBD assay detected both bacterial and human DNA at 20 fg, demonstrating higher analytical sensitivity than the real-time PCR method. Moreover, mock forensic samples were analyzed to confirm the assay applicability, and PCR inhibitor tolerance tests using humic acid and tannic acid were conducted to further validate their performance. Furthermore, the HBQ and HBD assays may be used in quality control processes for samples potentially affected by bacterial DNA or human mtDNA contamination and could also be applied to other fields such as food safety, environmental science, and biological research involving microbial DNA and human mtDNA.