PCR bias associated with conserved primer binding sites, used to determine genotype diversity within Citrus tristeza virus populations.
Citrus tristeza virus (CTV) is present in almost all of the major citrus production areas where it continues to reduce the profitability of citriculture. The accurate characterisation of CTV populations, which are usually made up of a number of disparate strains, requires the use of robust PCR protocols. Mismatches between primers and their corresponding binding sites may introduce primer-associated bias during amplification. The primer-associated bias of four sets of CTV specific primers, targeting the A and F regions and the p33 and p23 genes, were evaluated. This was done through the amplification of defined templates followed by their characterisation using the sequencing of multiple clones, as well as Illumina next generation sequencing. High levels of bias were found to be associated with the primer pairs targeting the A and F regions. The p33 gene primers were found to be biased against two genotypes and suggestions for preventing this apparent bias are discussed. The primer pair targeting the conserved p23 gene was found to have very little associated bias. Primers should undergo rigorous screening before being used to characterize virus populations that are known to exhibit high levels of variation, especially within primer binding sites.
Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa. Electronic address: david.read@fabi.up.ac.za.
David Alan
DA
Read
Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; Agricultural Research Council-Plant Protection Research Institute, Pretoria 0002, South Africa. Electronic address: gerhard.pietersen@up.ac.za.
Gerhard
G
Pietersen
D001665:Binding Sites; D002957:Citrus / Q000821:virology; D017790:Closterovirus / Q000235:genetics*; D017931:DNA Primers; D014644:Genetic Variation; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards
D017931:DNA Primers
Citrus tristeza virus; PCR bias; Viral populations