Accumulating Variation at Conserved Sites in Potyvirus Genomes Is Driven by Species Discovery and Affects Degenerate Primer Design

Abstract

Unknown and foreign viruses can be detected using degenerate primers targeted at conserved sites in the known viral gene sequences. Conserved sites are found by comparing sequences and so the usefulness of a set of primers depends crucially on how well the known sequences represent the target group including unknown sequences.Methodology/Principal FindingsWe developed a method for assessing the apparent stability of consensus sequences at sites over time using deposition dates from Genbank. We tested the method using 17 conserved sites in potyvirus genomes. The accumulation of knowledge of sequence variants over 20 years caused ‘consensus decay’ of the sites. Rates of decay were rapid at all sites but varied widely and as a result, the ranking of the most conserved sites changed. The discovery and reporting of sequences from previously unknown and distinct species, rather than from strains of known species, dominated the decay, indicating it was largely a sampling effect related to the progressive discovery of species, and recent virus mutation was probably only a minor contributing factor.Conclusion/SignificanceWe showed that in the past, the sampling bias has misled the choice of the most conserved target sites for genus specific degenerate primers. The history of sequence discoveries indicates primer designs should be updated regularly and provides an additional dimension for improving the design of degenerate primers.

PCR bias associated with conserved primer binding sites, used to determine genotype diversity within Citrus tristeza virus populations

“…Three of the CTV specific primer sets targeting conserved sites, namely the A and F (Rubio et al, 2001) and the p23 gene specific primers (Sambade et al, 2003), were designed more than a decade ago. It is therefore reasonable to assume that significant -consensus decay‖ (Zheng et al, 2008) has occurred due to the sequencing of additional CTV genomes.…”

“…When considering the additional 41 genomes (used as references in this study), an additional seven and nine nucleotide differences are collectively added to the binding 12 sites of the A and F regions respectively (data not shown). Zheng et al, (2008) quantified the potential for the consensus decay of conserved sites among members of Potyvirus. However, the loss of consensus among primer binding sites is often not considered when estimating viral population diversity.…”

“…These conserved sites can only be targeted by comparing known sequences of a specific virus or group of viruses. The efficacy of the resulting primers will be based on how well they correspond to the conserved sites of both the known and unknown sequences (Zheng et al, 2008). As the pool of known sequence information increases the conserved nature of target sites begins to degrade thorough process termed -consensus decay‖ (Zheng et al, 2008).…”

“…The efficacy of the resulting primers will be based on how well they correspond to the conserved sites of both the known and unknown sequences (Zheng et al, 2008). As the pool of known sequence information increases the conserved nature of target sites begins to degrade thorough process termed -consensus decay‖ (Zheng et al, 2008). Continued use of primers that no longer represent conserved sites may result in the preferential amplification of sequences from populations.…”

Molecular Evidence for Association of <I>Papaya ringspot virus</I> with Papaya from North East Hill Region of India

“…One step RT-PCR was performed in the same tube following standard method. The PCR reaction mixture (100 µl) contained 2 µl of primers (100 pmol/µl) NIb 2F and NIb 3R (Zheng et al, 2008), 5 units RNase inhibitor (Invitrogen), 10 units of reverse transcriptase (Qiagen Inc.), 5 units Taq polymerase (Qiagen Inc.), 1X PCR buffer, 10 mM dithiotheritol, 1XQs solution and 10 µM each of the dNTPs. The amplification conditions were: one cycle of 45 min at 42°C, 40 cycles of 30 s at 94°C, 2 min at 46°C and 1 min at 72°C and one cycle of 5 min at 72ºC.…”