There have been previous efforts to develop real-time PCR based BKV quantification assays [27, 28]. However, many of these assays were developed when the number of available DNA sequences in public databases was quite limited. Although multiple sequence alignments are routinely used to identify stable target regions for assay development, sequence variants have been observed in these regions as more strains and sequences become available. Therefore, when the number of isolates and sequences is limited, merely selecting a target region for assay development based on intraspecies nucleotide sequence alignments alone may not be sufficiently predictive of interstrain sequence conservation to result in a sensitive and specific assay.
We proposed that identifying areas of the genome that are conserved within and between species could be used to supplement limited nucleotide sequence information when designing assays. Target regions identified via standard intraspecies sequence alignment of full genome sequences from available isolates were further evaluated by interspecies amino acid analysis to select a single target region. The interspecies amino acid alignments assisted in narrowing down potential target regions by identifying areas containing functionally and/or structurally constrained sequences. Conservation in amino acid sequence among closely related viruses does not necessarily preclude the possibility of non-synonymous substitutions. Functional and structural constraints, however, do suggest that there would likely be fewer number of amino acid residues that can reside at that particular position than if the same region was under less stringent functional or structural constraint.
However, mutations in the nucleotide sequences giving rise to synonymous substitutions can potentially have equally adverse effects on the performance of a PCR assay. Possible mechanisms for constraints limiting synonymous mutations may include overlapping reading frames, especially if those overlapping regions are functionally or structurally important. Identifying potential cold spots in the genome that are less likely to undergo synonymous substitutions is a significant challenge and a major obstacle to developing a robust PCR-based viral assay. Intra-species nucleotide alignment of available BKV sequences was followed by detailed intra- and interspecies nucleotide analysis of the target region to aid in designing assay primers and probe.
In the present work, the utilization of the intraspecies nucleotide alignment of BKV with the intraspecies nucleotide alignment of a closely related virus, JCV, for which a significant number of sequences were available, proved to be useful in substantiating that the Polyoma_4.2(f) and the BKV-MGB probe binding regions within BKV was less likely to experience nucleotide sequence variation. The lack of polymorphisms at the corresponding positions in both BKV and JCV isolates provides stronger evidence for sequence conservation than does intra-species nucleotide analysis of BKV isolates alone. Conversely, the forward primer BKV_2.1(f) is reserved for use only as a secondary backup to Polyoma_4.2(f) due to the extent of polymorphisms seen among JCV isolates along the nucleotide stretch corresponding to the BKV_2.1(f) binding site.
Since the assay was first developed using ~160 BKV sequences, the number of available BKV complete genome sequences increased to 271 (Additional file 1). As presented in the results section, the increase in BKV sequence data did not demonstrate increased sequence variation in the region targeted by this analysis, validating the approach of incorporating the additional analyses discussed herein when designing a real-time PCR assay.
The amino acid and nucleotide sequence analyses that were performed identified the VP2 C-terminus as a probable functionally conserved region, consistent with the presence of an alpha-helix  and nuclear localization signal [19–22]. Targeting the VP2 gene for assay development is in contrast to the location of primers/probes for most existing BKV assays . Therefore this approach was successful in identifying a region different from those targeted by other assays that have become limited with the recognition of additional sequence variations.
Using functionally constrained regions for assay development might be expected to result in lack of specificity. However, sufficient nucleotide differences existed within the stable region to design an assay specific for BKV. The three nucleotide difference between the BKV and JCV at the probe binding site was sufficient to meet assay specificity requirements by utilizing a Taqman®-MGB probe. By having the probe bind to the anti-sense strand, the respective positions of the inter-species nucleotide differences between the BKV and JCV were such that the minor groove binder of the Taqman®-MGB probe could be ideally positioned to provide a high degree of specificity. This configuration also provided the added benefit of having the probe bind to the same strand (anti-sense) as the BK virus specific reverse primer rather than on the sense strand that binds the Polyoma_4.2(f) primer, which amplifies both JCV and BKV. It should be noted that the primer combination BKV_5.1(r) and Polyoma_4.2(f) is the only primer combination that has been designed and tested to perform under a BKV and JCV co-infection scenario.
Preliminary assay performance results indicate the BKV assay has a 6-log dynamic range, a lower detection limit of 1.5 × 101 copies/reaction and an intra-assay CV of ≤ 5.0%. Good precision is important for distinguishing between assay variation and assay results that are associated with clinical treatment or indicative of the need for treatment. A wide dynamic range is significant for clinical application of the assay since the clinical need is for identification of patients with high viral load who may be at immediate risk for developing BKVAN and also for identification of low viral loads in patients being monitored for BKV reactivation. The assay also does not detect JCV and the presence of JCV does not inhibit detection of BKV at the concentrations tested, suggesting that accurate detection and quantitative results for BKV will be obtained for BKV clinical samples that are co-infected with JCV.
The relative lack of sequence variants in the VP2 gene suggests that an assay targeting this region will permit accurate detection of BKV and estimates of viral load regardless of subtype (Additional file 2). Such information is important in identifying active BKV infections in transplant recipients in time to initiate appropriate treatment. Further testing of this BKV assay is needed to fully characterize assay performance characteristics, clinical performance and to evaluate the impact of sequence variations.