Abstract:
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.
