Metic Transplantation Laboratory, USC, Keck School of Medicine, Los Angeles, CA, USA.
Virol J. 2010 Oct 29;7:295. doi: 10.1186/1743-422X-7-295.
BK 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.
Employing 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%).
Identifying 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.
BK 病毒感染在免疫功能低下的个体中可能具有重要的临床意义。在某些情况下，建议检测和监测 BK 病毒的活性感染，因此开发了用于检测 BK 病毒的 PCR 检测方法。目前 BK PCR 检测方法的性能受到病毒多态性的限制，在检测方法开发时这些多态性未知，导致 BK 病毒的检测不一致。本研究的目的是确定 BK 病毒基因组的一个稳定区域，用于 PCR 检测，该区域受多态性的影响最小，因为随着 BK 病毒的更多序列数据的出现。
通过结合氨基酸和 DNA 序列比对以及种间分析等技术，在 BK 病毒 VP2 基因内鉴定到 BK 病毒基因组的一个保守、稳定的 PCR 靶区域。然后开发了一种实时定量 PCR 检测方法，该方法特异性针对 BK 病毒，分析灵敏度为 15 拷贝/反应（450 拷贝/ml），重复性高（CV≤5.0%）。
当可用的 DNA 序列数据有限时，通过结合多种分析技术阐明对基因组区域的潜在功能限制，可能有可能确定稳定的 PCR 靶区域。将这种方法应用于 BK 病毒的实时定量 PCR 检测方法的开发，产生了一种具有潜在临床应用和优于现有 BK 检测方法的准确方法。
