Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology.
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.
Zwart
Mark P
MP
Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.
van Oers
Monique M
MM
Cory
Jenny S
JS
van Lent
Jan W M
JW
van der Werf
Wopke
W
Vlak
Just M
JM
D000818:Animals; D052160:Benzothiazoles; D044822:Biodiversity; D003959:Diamines; D005838:Genotype; D007814:Larva / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D018411:Spodoptera / Q000821:virology
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I