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 a...

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Bibliographic Details
Published in:	Biotechnology and bioengineering Vol. 96; no. 4; pp. 810 - 814
Main Authors:	Hitchman, Richard B., Siaterli, Evangelia A., Nixon, Clare P., King, Linda A.
Format:	Journal Article
Language:	English
Published:	Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-03-2007 Wiley Wiley Subscription Services, Inc
Subjects:	Baculoviridae - genetics Baculovirus Biochemistry Biological and medical sciences Biotechnology Biotechnology - methods DNA Primers Fundamental and applied biological sciences. Psychology Genetic recombination high throughput Molecular biology Polymerase Chain Reaction - methods quantitative PCR (QPCR) Recombinant Proteins - biosynthesis Sensitivity and Specificity titer Titrimetry Viral Plaque Assay Viruses Virus Polymerase chain reaction baculovirus Baculoviridae Recombinant virus titer Real time high throughput quantitative PCR (QPCR) Quantitative analysis
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Summary:	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 CT 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. Biotechnol. Bioeng. 2007;96:810–814. © 2006 Wiley Periodicals, Inc.
Bibliography:	South East Proof of Concept funding (SEPOC) The Biotechnology and Biological Sciences Research Council (BBSRC) istex:83FF4217EF9B64294BD8D55362FFB0C30690E02E ArticleID:BIT21177 ark:/67375/WNG-BS7KZNQW-6 Richard B. Hitchman and Evangelia A. Siaterli contributed equally to this work. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Feature-1 SourceType-Other Sources-1 content type line 63 ObjectType-Correspondence-1 ObjectType-Undefined-2 ObjectType-Article-3
ISSN:	0006-3592 1097-0290
DOI:	10.1002/bit.21177