Abstract
BackgroundBK virus infections can have clinically significant consequences in immunocompromised individuals. Detection and monitoring of active BK virus infections in certain situations is recommended and therefore PCR assays for detection of BK virus have been developed. The performance of current BK PCR detection assays is limited by the existence of viral polymorphisms, unknown at the time of assay development, resulting in inconsistent detection of BK virus. The objective of this study was to identify a stable region of the BK viral genome for detection by PCR that would be minimally affected by polymorphisms as more sequence data for BK virus becomes available.ResultsEmploying a combination of techniques, including amino acid and DNA sequence alignment and interspecies analysis, a conserved, stable PCR target region of the BK viral genomic region was identified within the VP2 gene. A real-time quantitative PCR assay was then developed that is specific for BK virus, has an analytical sensitivity of 15 copies/reaction (450 copies/ml) and is highly reproducible (CV ≤ 5.0%).ConclusionIdentifying stable PCR target regions when limited DNA sequence data is available may be possible by combining multiple analysis techniques to elucidate potential functional constraints on genomic regions. Applying this approach to the development of a real-time quantitative PCR assay for BK virus resulted in an accurate method with potential clinical applications and advantages over existing BK assays.
Highlights
BK virus infections can have clinically significant consequences in immunocompromised individuals
We report that interspecies amino acid and nucleotide sequence analysis, in conjunction with intraspecies nucleotide sequence alignment, can elucidate genomic regions that may be under potential functional constraints and that these regions can be targeted for primer and probe design to improve assay performance
These analyses show that sequence variants are widespread throughout the BK virus (BKV) genome and occur in all 6 viral genes
Summary
BK virus infections can have clinically significant consequences in immunocompromised individuals. BKV and JCV are ubiquitous in human populations worldwide with a seroprevalence in adults of 70%-80% [1,2,3,4] They establish persistent, latent infections and are capable of reactivating in immunosuppressed hosts [5,6,7]. Primers and probes developed for quantitative BKV testing based on limited available sequence data from few viral isolates suffer reduced performance in detection of viral isolates with sequence variations in these regions. This in turn can lead to inconsistent detection of virus, inaccurate quantitation of viral load and difficulty comparing results between assays
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