Year: 2024 | Month: February | Volume 14 | Issue 1
Development of gp64 Gene based Real-time Quantitative PCR Assay for Rapid and Accurate Determination of Baculovirus Titer
T. Rama
Mohini Saini
Lekshmi S. Rajan
Deepika Bisht
Ram Bachan
Praveen K. Gupta
DOI:10.30954/2277-940X.01.2024.12
Abstract:
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.
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