[An efficient and accurate method for counting target molecules in phage-display peptide library]

Area of Science:

• Microbiology
• Molecular Biology
• Virology

Context:

• Accurate bacteriophage titer determination is crucial for various applications, including phage therapy and molecular biology research.
• Traditional methods like the double-layer agar plate (DLAP) assay are time-consuming and can exhibit significant variability.
• Developing rapid and reliable methods for phage quantification is essential for efficient research and application.

Purpose:

• To compare the accuracy, efficiency, and variability of real-time PCR assay versus the double-layer agar plate (DLAP) method for determining bacteriophage titer.
• To evaluate the suitability of both methods for quantifying low, intermediate, and high phage concentrations.

Summary:

• Both DLAP and real-time PCR accurately measured bacteriophage titer across different concentrations.
• The DLAP method required multiple replicates (10) with larger sample volumes (10 microliters) and showed higher within-assay coefficient variation (CV) ranging from 4.93% to 30.38%.
• Real-time PCR required fewer replicates (3) with smaller sample volumes (1 microliter) and demonstrated significantly lower within-assay CV (0.02% to 0.25%), indicating superior precision and efficiency.

Impact:

• Real-time PCR presents a more efficient, rapid, and precise alternative to DLAP for bacteriophage titer determination.
• This advancement can accelerate research in phage biology, diagnostics, and therapeutic applications by enabling quicker and more reliable phage quantification.
• The findings support the adoption of real-time PCR for routine bacteriophage quantification in laboratory settings.