Digital PCR (dPCR) SOP for quantification of antibiotic resistance genes

Authors: Condon, Justine; Durso, Lisa

Submitted to: Protocols.io
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2025
Publication Date: 4/4/2025
Citation: Condon, J.C., Durso, L.M. 0202. Digital PCR (dPCR) SOP for quantification of antibiotic resistance genes. Protocols.io. https://doi.org/10.17504/protocols.io.5qpvo96wzv4o/v1.
DOI: https://doi.org/10.17504/protocols.io.5qpvo96wzv4o/v1

Interpretive Summary: This protocol describes an assay designed to detect four specific targets related to antibiotic resistance – which is when infectious disease causing organisms that used to be easily treated with antibiotics no longer work.  This means patients are sick longer, or in extreme instances, that the infection cannot be cured.  It uses a method called digital PCR (dPCR).  dPCR is a tool that allows for very specific detection and enumeration of bacterial targets.  It works by taking thousands of measurements on a single sample.  Because of the high number of measurements for each sample, the method is very robust, and provides exceptionally accurate counts of the target.

Technical Abstract: Purpose To describe the correct procedures on how to set-up digital polymerase chain reaction (dPCR), for selected antibiotic resistance genes and determinants: sulfonamide (sul), erythromycin (erm(B)), extended spectrum ß-lactamase (blaCTX-M-1), integrase (intI1), and tetracycline tet(A), tet(O), and tet(X). Scope Digital PCR is a specialized approach to nucleic acid detection and quantification that estimates absolute numbers of molecules through statistical methods. Digital PCR (dPCR) is the third-generation PCR technique. It sequesters probes and nucleic acids, and uses the same fundamental chemistry as qPCR, but unlike qPCR data, dPCR data are collected at the endpoint of the reaction mix. Before amplification, a bulk PCR reaction made up of nucleic acid, primers, probes, and master mix is digitized into many thousands of nanoliter-sized microreactions. As this digitization process distributes the PCR mix across so many microreactions, each microreaction will effectively either contain one, zero, or just a handful of the target nucleic acid molecules (ThermoFisher, 2015). The isolated microreactions are then amplified, and data are collected from each microreaction at the end of the thermal cycling process. Microreactions that do not contain the target will not show post-amplification fluorescence, while those that do contain the target will show post-amplification fluorescence (ThermoFisher, 2015). While the concept of reading the fluorescent signals from amplified genes is the same compared to qPCR, dPCR is better at preserving the initial reaction mix conditions and clearly representing amplification progress, minimizing the occurrence of primer-dimers, and false amplification reading (Park, 2021). The QIAcuity Digital PCR System is designed to deliver precise and multiplexed quantification results for mutation detection, copy number variation (CNV), gene expression studies, gene-editing analysis, and many more. This nanoplate-based system seamlessly integrates a standard dPCR workflow of partitioning, thermocycling and imaging into a walk-away automated platform with minimal hands-on time (Qiagen, 2024).