A rapid and accurate method for determining titers of baculovirus for protein expression

The Baculovirus Expression Vector System (BEVS) is extensively used for expressing target proteins, necessitating accurate baculovirus titration to ensure the output. Traditional plaque assay assisted titration, while reliable, is labor-intensive and time consuming. Here, we introduce an improved quantitative real-time polymerase chain reaction (qPCR)-based method for determining baculovirus titers. Briefly, target-gene specific primers are designed to produce a 180-bp PCR product using viral DNA as the template. The PCR products are purified and the mass weight of the PCR products are quantified based on 260-nm absorbance. Based on the molecular weight of the 180bp PCR product, the mass weight is then converted to the molar concentration which is finally converted to molecule number by applying the Avogadro constant. Such PCR product is serially diluted and used as the template for qPCR. The qPCR Ct values between serial dilutions are used to generate the standard curve. Finally, the Avogadro’s number (i.e. titer) of the test viral DNA is inferred from its qPCR Ct value by comparing with the standard curve. Our method eliminates the need for a pre-titrated viral stock, reducing variability caused by storage conditions. This advancement is particularly beneficial for large-scale production of the target protein using the BEVS.