Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology

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Abstract

Two bacmid-derived Autographa californica Multiple-capsid Nucleopolyhedrovirus genotypes ¿ that differ only in a short tag sequence for differential PCR recognition ¿ were generated. By electron microscopy, these genotypes were found to have identical polyhedra morphology. Mixtures of quantified polyhedra were made and used to validate a SYBR Green I-based quantitative real-time PCR (qPCR) to determine genotype frequencies in mixed genotype populations. The PCR could accurately quantify genotype ratios over a range of 8 orders of magnitude. Only a small correction of the genotype ratio was necessary to obtain a valid result. Low levels of aspecific background (a fluorescent signal when the template corresponding with the primer set used is not present) were measured in these validation experiments and in a typical laboratory setup. A small fitness difference between the genotypes generated was observed in a median lethal dose bioassay. The bacmid-derived virus genotypes generated and the qPCR assays are valuable tools for studying the population biology of baculoviruses.
Original languageEnglish
Pages (from-to)146-154
JournalJournal of Virological Methods
Volume148
Issue number1-2
DOIs
Publication statusPublished - 2008

Keywords

  • nuclear polyhedrosis-virus
  • polymerase-chain-reaction
  • trichoplusia-ni
  • sybr-green
  • mosaic-virus
  • wild-type
  • rt-pcr
  • nucleopolyhedrovirus
  • mutants
  • larvae

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