Digital PCR modeling for maximal sensitivity, dynamic range and measurement precision

PLoS One. 2015 Mar 25;10(3):e0118833. doi: 10.1371/journal.pone.0118833. eCollection 2015.

Abstract

The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for genetic quantification. A challenge associated with digital PCR experiments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (Po) is achieved by targeting ~1.59 mean copies per partition (λ), and that dynamic range (R) includes the space spanning one positive (λL) to one negative (λU) result from the total number of partitions (n), these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next considered. The resultant effects on sensitivity and precision are established via Monte Carlo simulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the framework is presented enabling detection across 3.33 logs of starting copy concentration.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Models, Theoretical*
  • Monte Carlo Method
  • Polymerase Chain Reaction / instrumentation*
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity

Grants and funding

All the funding and sources of support are from Life Technologies, now a part of Thermofisher Scientific. The funder provided support in the form of salaries for all authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.