A Droplet Digital PCR Assay for Quantification of Bacteriophage Viral Vector Titer and Purity.

Purpose: Bacteriophage (phage) based vectors offer considerable promise as tools for tuning the microbiome with molecular and genetic precision. However, standardized methods to rigorously characterize phage vectors remain lacking. Here, we present an optimized digital droplet PCR (ddPCR)-based assay for quantifying both the purity and potency of phage vector preparations. Methods: We utilized central composite design to develop a ddPCR assay capable of quantifying the number of phage vector capsids packed with the phage vector genome or packed with the transgenic DNA of interest. This assay targets 2 unique DNA barcodes, designed to be biologically inert and maximally orthogonal to existing DNA sequences. Results: Through stringent optimization, we were able to achieve assay conditions that enable a dynamic range of nearly 3 orders of magnitude and correct for systemic error in the assay. We then show that biological activity assays consistently underestimate transgene-packed vectors titers, leading to overestimation of true transduction efficiency, particularly when contamination by genome-packed vectors is high. We further demonstrate how this approach facilitates optimization of vector production conditions and substantially improves the precision and reproducibility of phage vector transduction. Conclusion: Compared to assays of biological activity, this optimized ddPCR assay has improved accuracy and, through design of experiments optimization, high precision (CVs = 5.5 ± 1.3% and 4.5 ± 1.0% for the genome and transgene barcodes, respectively). This assay can be broadly adopted to characterize and quality control vector preparations for various applications.