A Quick and Efficient Method to Quantify Baculo Virus by Quantitative Real Time PCR

Mahammad Azhar^1*, R. Somashekhar² and P.P.B.S. Kumar³

1Department of Biotechnology, Jawaharlal Nehru Technological University, Hyderabad - 500 072, India. 2Department of Biotechnology, Azyme Biosciences, Bangalore - 560 069, India. 3Department of Biotechnology, Acharya Nagarjuna University, Guntur - 522 510, India.

DOI : http://dx.doi.org/10.13005/bbra/1178

ABSTRACT:

Recombinant protein expression using the Baculovirus Expression System is widely used for the production of therapeutics, diagnostics, and reagents. The current 'gold standard' for Baculovirus titration, the plaque assay, is labor intensive and time consuming. The real-time PCR assay has the advantage of speed and accuracy. We determined Baculovirus tire by a novel quantitative PCR (qPCR) method, using Baculovirus gene gp64 (Coat protein) specific primers and a recombinant gene-specific primers. Virus specific amplification and gene specific amplification were compared, and both values were found to be very close, suggesting that most of the virus were expressing our gene of interest. This method is useful to determine the percentage of non-recombinant virus particles in virus solution. Further, we compared real-time PCR results with plaque assay, and found that the values were much similar. This method has the advantage of time saving as it completes in 2-3 hours, and not only quantifies the virus, but also quantify the virus, containing the gene of interest.

KEYWORDS: QPCR; Baculovirus; AcMNPV; MOI; gp64

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