Virometrics

Vector Shedding

Viral Titer

qPCR Quantitation

Design of Experiments

Transgene vector as qPCR template.
Primers/probe design based on transgene sequence by using NCBI primer/probe design tool.
Confirm aplicon size be gel electrophoresis.
Reference gene selection for duplex PCR reactions.

Test standard curve with 8 calibrators. Make 8 calibrators: spiking transgene vector into 1ug genomic DNA: 1x107, 1x106, 1x105, 1x104, 1x103, 1x102, 10, 1 copies/mL and blank.
Amplification efficiency (90%-110%): Regression R2>0.99
Duplex PCR reactions with reference gene qPCR in the same reaction.

Program Optimization

Gradient qPCR test determine the optimal Tm.

qPCR amplification efficiency.
Assay sensitivity: lower limit of quantification and upper limit of quantification.
Assay accuracy and precision.
Assay specificity.
Assay selectivity.
Assay dilutional linearity.
Assay stability.

Quality Control

Transgene vector purity and concentration.
Matrix genomic DNA purity and concentration.
Sample DNA purity and concentration.
qPCR assay validation parameters and criteria.

ddPCR Quantitation

ddPCR (droplet digital polymerase chain reaction) assays work by partitioning PCR reactions within oil droplets.
Each PCR reaction is allowed to run to completion prior to analysis.
Taq-Man probes or EvaGreen dye provides a correlation between PCR amplification and flourescence

10,000 – 20,000 droplets are produced in each sample.
The fluorescence intensity is measured for each droplet in each sample.
Each droplet is treated as a binary value.
Droplets above the fluorescent threshold are considered positive.
Droplets below the fluorescent threshold are considered negative.

The fraction of negative droplets are derived from the droplet fluorescence.
An absolute DNA concentration is calculated from this fraction of negative droplets using a Poisson distribution.

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