Validation of Countable PCR reproducibility across technical replicates, users, and systems

Introduction

Precise DNA quantification is crucial for applications such as cell and gene therapy manufacturing, copy number determination, rare event detection, and general quantification of gene targets. Although digital PCR is widely used for DNA quantification, it has technical limitations that hinder low-variability results, typically yielding a coefficient of variation (CV) of around 10% [1]. These include:

1. Reliance on Poisson correction, which depends on consistent and accurate partition volumes to calculate copy number reliably [2, 3, 4].
2. A limited number of partitions, which begin to saturate at target counts typically near 100,000 molecules. As the target molecule counts approach the upper limit of the dynamic range, an increasing number of partitions contain multiple molecules, leading to reduced measurement resolution and accuracy [5].
3. Manual thresholding, which introduces user-dependent bias and reduces reproducibility.

Countable PCR partitions individual target molecules within a 3D gel-like matrix, enabling direct counting across four channels up to 1 million counts. With over 30 million partitions ensuring single occupancy, the need for Poisson correction is eliminated, and the dependence on partition consistency and high occupancy correction is removed. In addition, Countable PCR’s light sheet imaging and automated counting algorithm allow for high-throughput processing with reduced variability between replicates and minimal user-dependent error.

Here, we demonstrate the reproducibility of Countable PCR across a six-log dynamic range and different users and instruments. These results highlight the power of direct counting to deliver low-variability data and consistent performance, which is essential for cross-validation and reliable assay development at multiple sites.