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

Bacteriophage (phage) vectors, bacterial viruses engineered to deliver specific transgenes into target bacteria, offer considerable promise as tools for tuning the microbiome. However, the field currently lacks standardized methods for characterizing phage vectors with the same rigor as those for viral vectors used in human gene therapy. To develop a robust and reproducible viral vector system, it is critical to measure both transduction efficiency and the true dose of vector particles used. Here, we use central composite design to develop a digital droplet PCR (ddPCR) assay for precise quantification of transgene and vector genome-packed phage vector titers following an established production process. This duplex assay uses probes specific to 2 unique DNA barcodes inserted into the vector genome and transgene cassette, enabling precise quantitation of each population with a dynamic range of nearly 3 orders of magnitude. We show biological activity assays can consistently underestimate the number of transgene-packed vectors, potentially greatly overestimating the true transduction efficiency, particularly when contamination by genome-packed vectors is high. This ddPCR approach can be broadly adopted to characterize phage particles for a variety of biomedical applications. ### Competing Interest Statement The authors have declared no competing interest.