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Free DNA partially clarifies discrepancies between qPCR and the conventional phage quantification method
Saar Van Overfelt1,2, Hans Duyvejonck1,2, Femke Baeke3,4
1Research Centre Health and Water Technology, University of Applied Sciences and Arts Ghent, Ghent, Belgium.
Plos One
|December 3, 2024
Summary
Accurate phage quantification is crucial for therapy and industry. DNase treatment before qPCR reduces overestimation of infectious phage particles, improving accuracy, though storage time impacts results.
Area of Science:
- Microbiology
- Molecular Biology
Background:
- Accurate phage quantification is essential for therapeutic and industrial applications.
- The double agar overlay (DAO) method is accurate but laborious; quantitative PCR (qPCR) is faster but overestimates phage particles.
Purpose of the Study:
- To improve the accuracy of phage quantification using qPCR by incorporating DNase treatment.
- To assess the impact of DNase treatment on Staphylococcus aureus phage ISP quantification.
Main Methods:
- Phage quantification using qPCR with and without prior DNase treatment.
- Comparison of qPCR results with the gold standard double agar overlay (DAO) method.
- Negative contrast immune electron microscopy to detect free DNA.
Main Results:
- DNase treatment significantly decreased phage concentration measured by qPCR.
- Discrepancies between qPCR and DAO methods persisted for some phage stocks.
- The accuracy of qPCR quantification was influenced by the storage period of the phage stock, with older stocks showing greater discrepancies.
Conclusions:
- DNase treatment enhances the accuracy of qPCR for quantifying infectious phage particles.
- Storage duration affects the reliability of qPCR phage quantification.
- Further optimization is needed to fully align qPCR with DAO method accuracy for all phage stocks.
