Development of a multiplex ddPCR assay for simultaneous absolute quantification of bacterial, fungal, and human DNA

Molecular methods in clinical and research applications frequently encounter complex mixtures of human and microbial DNA, sometimes alongside environmental or reagent contaminants. In metagenomic studies, the presence of host contamination poses a significant challenge, reducing the assay sensitivity of microbial detection and characterization. Different host-depletion and microbial enrichment platforms have been developed to reduce or eliminate host DNA in samples predominantly composed of human DNA. To establish an effective method to assess the efficacy of host DNA depletion or microbial enrichment platforms, this study aimed to develop a multiplex, broad-range 16S/18S ribosomal DNA droplet digital PCR (rDNA ddPCR) assay capable of simultaneously quantifying bacterial and fungal DNA, along with a human housekeeping gene, RPP30 (Ribonuclease P/MRP Subunit P30). Genomic DNA from key representatives of Gram-positive bacteria, Gram-negative bacteria, and fungi was tested in broad-range 16S/18S rDNA duplex (16S/RPP30 or 18S/RPP30) and triplex (16S/18S/RPP30) ddPCR assays to determine optimal assay conditions, specificity, and sensitivity. This assay demonstrated high sensitivity, specificity, and reproducibility, with detection limits of approximately 3 copies/µL for the 16S target (0.5 pg Staphylococcus aureus gDNA) and 1-2 copies/µL for the 18S target (16 fg of Candida albicans gDNA) in both duplex and triplex formats. Within a defined range, a linear relationship was observed between microbial DNA input and 16S/18S rDNA copy number by ddPCR. Furthermore, different commercial ddPCR master mixes had contrasting effects on the amplitudes of positive 16S/18S droplet clusters. As a proof of concept for the assay's utility in metagenomic studies, we demonstrated that one extraction kit achieved more efficient depletion of human DNA and better enrichment of microbial DNA. In summary, we developed a multiplex, broad-range 16S/18S ddPCR assay with high sensitivity and specificity, which holds promise as a QA/QC (Quality Assurance/Quality Control) platform in metagenomic studies and other research settings.