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Updated: Aug 5, 2026
Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction
Published on: October 11, 2024
Rapid and quantitative measurement of bacteriophage infectivity via fully automated droplet digital PCR
Yanfei Liu1,2, Huiwei Zhao3,4, Xinyue Cao5,6
1Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Shenyang, Liaoning, China.
Abstract:
The clinical translation of phage therapy for multidrug-resistant infections is constrained by the lack of rapid, standardized therapeutic phage selection. Here, we introduce digital phage susceptibility testing (dPhaST), an automated droplet digital PCR workflow that quantifies phage-induced DNA release as a molecular signature of lysis. By targeting conserved 16S rRNA regions, dPhaST measures lytic activity across diverse bacterial pathogens within 3 h. Across 122 phage-host combinations involving 19 bacterial strains from six species, dPhaST shows 95.9% concordance with spot tests while resolving weak and heterogeneous lytic activities that are not readily distinguished phenotypically. It remains robust during the early infection window despite phage-encoded nuclease activity and tolerates phage cross-contamination better than spot tests. The method captures defense-mediated interactions involving CRISPR-Cas and Sir2-HerA systems. In this work, we show that automated digital quantification enables rapid and mechanistically informative profiling of early phage lytic efficacy across Gram-positive and Gram-negative pathogens.
Insights
Digital phage susceptibility testing (dPhaST) rapidly quantifies phage efficacy against multidrug-resistant infections. This automated method measures DNA release to identify effective therapeutic phages within hours, improving treatment selection.
Area of Science:
- Microbiology
- Molecular Biology
- Bioinformatics
Background:
- Phage therapy offers a promising alternative for treating multidrug-resistant infections.
- Clinical translation is hindered by the absence of rapid, standardized methods for selecting effective therapeutic phages.
- Current methods for assessing phage efficacy are often slow and lack sensitivity for heterogeneous responses.
Purpose of the Study:
- To introduce digital phage susceptibility testing (dPhaST), an automated workflow for rapid and accurate quantification of phage lytic activity.
- To establish dPhaST as a reliable method for selecting therapeutic phages against diverse bacterial pathogens.
- To characterize phage-host interactions and bacterial defense mechanisms using dPhaST.
Main Methods:
- Development of an automated droplet digital PCR (ddPCR) workflow (dPhaST).
- Quantification of phage-induced bacterial DNA release as a marker of lysis.
- Targeting conserved 16S rRNA bacterial genes for broad pathogen detection.
- Comparison of dPhaST results with traditional spot tests across numerous phage-host combinations.
Main Results:
- dPhaST accurately quantifies phage lytic activity within 3 hours across diverse bacterial pathogens.
- Achieved 95.9% concordance with spot tests, while also resolving weak or heterogeneous lysis.
- Demonstrated robustness in early infection stages and tolerance to phage cross-contamination.
- Successfully captured phage interactions with bacterial defense systems like CRISPR-Cas.
Conclusions:
- Automated digital quantification via dPhaST enables rapid and mechanistically informative profiling of phage lytic efficacy.
- dPhaST facilitates faster and more precise selection of therapeutic phages for multidrug-resistant infections.
- This method holds significant potential for advancing phage therapy clinical translation across Gram-positive and Gram-negative pathogens.
