Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles.
We describe the use of quantitative PCR (QPCR) to titer recombinant baculoviruses. Custom primers and probe were designed to gp64 and used to calculate a standard curve of QPCR derived titers from dilutions of a previously titrated baculovirus stock. Each dilution was titrated by both plaque assay and QPCR, producing a consistent and reproducible inverse relationship between C(T) and plaque forming units per milliliter. No significant difference was observed between titers produced by QPCR and plaque assay for 12 recombinant viruses, confirming the validity of this technique as a rapid and accurate method of baculovirus titration.
Hitchman
Richard B
RB
School of Biological & Molecular Sciences, Headington Campus, Oxford Brookes University, Oxford OX3 0BP, UK. rhitchman@brookes.ac.uk
Siaterli
Evangelia A
EA
Nixon
Clare P
CP
King
Linda A
LA
D016367:Baculoviridae / Q000235:genetics*; D001709:Biotechnology / Q000379:methods; D017931:DNA Primers; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012680:Sensitivity and Specificity; D017186:Titrimetry; D010948:Viral Plaque Assay
D017931:DNA Primers; D011994:Recombinant Proteins