Quantifying the Replication Efficiency of Wild-Type and Mutant Viruses

Begin with a qPCR plate containing a master mix with DNA polymerase and nucleotides.

Add viral cDNA derived from the culture supernatant of blood immune cells co-infected with wild-type and mutant viral strains.

Next, add serially diluted plasmid DNA with known copy numbers as standards.

Add strain-specific primers and a probe with a fluorescent reporter and a quencher to the corresponding wells.

Place the plate in the instrument and run the PCR program.

The high temperature activates the polymerase and denatures the cDNA.

As the temperature decreases, the primers and probes bind to their complementary sequences.

The polymerase extends the DNA and cleaves the probe, separating the reporter from the quencher, generating fluorescence that increases with each cycle.

Plot the fluorescence data to determine the Ct value, the cycle at which fluorescence crosses the detection threshold.

A higher Ct value for the mutant than for the wild-type strain indicates a lower initial cDNA copy number, representing reduced replication efficiency.

For qPCR, prepare a standard serial dilution series in triplicate of the plasmid pNL4-3VifA diluting tenfold in each step, from three times 10 to the six copies per