Application of real-time PCR to significantly reduce the time to obtain recombinant MVA virus

•Novel single-tube real-time PCR to determine a ratio of wild type and rMVA in plaques.•Rapid generation of new pure rMVA in 11 days.•Plaque purification is less effective then end-point dilution as the last purification step.•Various marker gene selected plaques differ greatly in proportion of rMVA...

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Bibliographic Details
Published inJournal of virological methods Vol. 289; p. 114056
Main Authors Orlova, O.V., Glazkova, D.V., Tsyganova, G.M., Antoshkina, I.V., Mintaev, R.R., Tikhonov, A.S., Bogoslovskaya, E.V., Shipulin, G.A.
Format Journal Article
LanguageEnglish
Published Netherlands Elsevier B.V 01.03.2021
Subjects
Animals
Antigens, Viral
Cell Line
Genetic Vectors
Humans
MVA
Real-time PCR
Real-Time Polymerase Chain Reaction
SARS-CoV-2
SARS-CoV-2 - genetics
Vaccine
Vaccinia virus - isolation & purification
MVA
SARS-CoV-2
Vaccine
Real-time PCR
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Summary:•Novel single-tube real-time PCR to determine a ratio of wild type and rMVA in plaques.•Rapid generation of new pure rMVA in 11 days.•Plaque purification is less effective then end-point dilution as the last purification step.•Various marker gene selected plaques differ greatly in proportion of rMVA. Obtaining a pure recombinant Modified Vaccinia Ankara (MVA) virus is a multistage, time-consuming procedure. We describe a novel single-tube real-time PCR which enables determination of the amount of wild type and recombinant viruses and their ratio in plaques. Use of the real-time PCR significantly reduce the time and efforts needed to obtain purified recombinant MVA. The new approach has been applied to generate recombinant MVAs encoding different SARS-COV-2 antigens.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
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ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2020.114056