Development of gp64 gene based real-time quantitative PCR assay for rapid and accurate determination of baculovirus titer

The present study was conducted to develop a simple and rapid quantitative real-time PCR (qPCR) assay for determination of baculovirus titer. Recombinant baculovirus expressing CE1E2 structural proteins of classical swine fever virus (CSFV) along with green fluorescent protein (GFP) reporter developed in our lab was used for the study. Sf21 cells were infected with tenfold dilution (10⁻¹ to 10⁻¹⁰) of baculovirus stock and GFP fluorescence was visualized. The titer (5.75 × 10⁷ TCID₅₀/mL) calculated by Reed and Muench method was taken as a standard stock to develop qPCR. DNA was isolated from baculovirus stock and checked for amplification of 175 bp baculovirus gp64 by standard PCR. Different dilutions of isolated DNA (10⁻¹ to 10⁻⁷) from P3 baculovirus stock were used as template and gp64 primers were used to determine the titer by qPCR. A linear relationship was obtained from 10⁰ to 10⁶ TCID₅₀ per 100 µL (Y = −3.34 X + 40.19, r² = 0.97). Using this equation, titer of unknown recombinant baculovirus stock was calculated to be 5.7 × 1 0⁷ per mL. Intra and inter assay coefficient of variations for qPCR results were less than 5%. The titration of baculovirus by this qPCR assay can be completed within 2–3 hrs compared to 10–12 days in the end point dilution method. To conclude, SYBR Green based qPCR titer estimation is a reliable, rapid and accurate assay for the titration of baculovirus and comparable to traditional end point dilution method.