Abstract
We introduce Countable PCR, a novel platform that enables transgene integrity and viral titer characterization, including long-read amplicons, within a single assay, with high sensitivity and low variability.
Current methodologies in the field present notable drawbacks for providing reliable viral titer measurements: quantitative, or real-time PCR (qPCR or rt-PCR, respectively) is too variable; digital PCR (dPCR) suffers from a narrow dynamic range, high Poisson errors, and arbitrary thresholding. Further, it is highly challenging to conduct both titer and genome integrity assessments in a single assay, as dPCR has partitioning limitations. Countable PCR overcomes these challenges by directly counting single molecules.
Here, we demonstrate the value of direct molecule counting by designing a novel multiplex assay that interrogates different regions of the gene-ofinterest (GoI) or region-of-interest (RoI) to accurately quantify the ratio of partial vs. complete transgene while simultaneously measuring titer in the same sample. Unlike dPCR, this single-molecule approach prevents false cooccupancy of fragmented, unlinked targets, ensuring accurate genome integrity data. Complete transgene integration is further validated with the ability to analyze long-read amplicons, which we demonstrate here up to 2000 bp. Together, this work demonstrates how Countable PCR makes gene therapy viral characterization workflows more versatile and approachable compared to other methods.
Download
