Recombinant Virus Quantification Using Single-Cell Droplet Digital PCR: A Method for Infectious Titer Quantification

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Accurate viral quantification is crucial for research and clinical diagnostics.
• Existing RNA virus quantification methods have limitations, including inhibitor sensitivity and the need for standard curves.
• Recombinant Semliki Forest virus (SFV) vectors are valuable tools in molecular biology and gene therapy.

Purpose of the Study:

• To develop and validate a droplet digital PCR (ddPCR) method for quantifying recombinant, replication-deficient SFV vectors.
• To assess the stability and reproducibility of the ddPCR method using different primer sets.
• To establish a single-cell ddPCR approach for quantifying infectious virus units and infected cells.

Main Methods:

• Utilized droplet digital PCR (ddPCR) for precise quantification of SFV vector genomes.
• Optimized ddPCR conditions, including primer selection (targeting transgenes, nsP1, and nsP4 genes) and annealing/extension temperatures.
• Developed a single-cell ddPCR assay by including infected cells in the PCR mixture, normalizing with β-actin primers.

Main Results:

• The ddPCR method demonstrated stability and reproducibility for quantifying SFV vector genomes.
• Successfully measured genome titers of mixed populations of replication-deficient recombinant virus particles.
• Quantified both the number of infected cells and virus infectious units using the single-cell ddPCR approach.

Conclusions:

• Droplet digital PCR (ddPCR) provides a robust and reproducible method for quantifying recombinant SFV vectors.
• The developed single-cell ddPCR assay enables accurate measurement of infectious units and infected cells.
• The single-cell ddPCR approach holds potential for clinical applications in quantifying infected cells.