Department of Pathology, Children's Medical Center, Dallas, TX, USA.
Mol Cell Probes. 2013 Oct-Dec;27(5-6):230-6. doi: 10.1016/j.mcp.2013.08.001. Epub 2013 Aug 20.
Several studies have shown that BK viral load in plasma and urine are reliable markers for the detection of BK virus associated nephropathy (BKVAN) in renal transplant patients. We developed a quantitative real time PCR assay based on TaqMan technology for the measurement of BK viral load in plasma and urine. Considering the high similarity of the nucleotide sequence of the BK virus (BKV) with the JC virus (JCV), we designed this assay to specifically amplify BKV. We determined the viral DNA recovery rate on manual (QIAGEN's QIAamp DNA Blood Mini Kit) and automated (BioMerieux's NucliSENS EasyMAG) extraction methods. The comparison showed a higher viral DNA recovery rate on the automated extraction (61-76% in plasma and 52-65% in urine) as compared to the manual method (49-52% in plasma and 33-56% in urine). Quantitation of the viral load was performed using an external standard curve that was constructed with serial dilution of a plasmid containing the full length of the BKV genome. Commercially available quantitative BKV standards showed good correlation with the plasmid standard. The reproducibility of the assay was determined based on the Ct values of the amplified products as well as in BK copies per milliliter of sample. This assay is linear over a 7 log range (10 to 1 × 10(7) copies per reaction), no cross-reactivity was detected with the closest-related polyomavirus JCV, as well as other viruses that may be found in immunocompromised patients, and human genomic DNA. The limit of detection of the assay is 300 copies per milliliter in both plasma and urine and the limit of quantitation is 1000 copies per milliliter using the NATtrol BK Virus Linearity Panel (ZeptoMetrix). This real time PCR assay provides a reliable and sensitive method for the quantitation of BKV in plasma and urine samples.
几项研究表明，血浆和尿液中的 BK 病毒载量是检测肾移植患者 BK 病毒相关性肾病（BKVAN）的可靠标志物。我们开发了一种基于 TaqMan 技术的定量实时 PCR 检测方法，用于测量血浆和尿液中的 BK 病毒载量。考虑到 BK 病毒（BKV）与 JC 病毒（JCV）的核苷酸序列高度相似，我们设计了该检测方法以特异性扩增 BKV。我们确定了手动（QIAGEN 的 QIAamp DNA 血液迷你试剂盒）和自动（BioMerieux 的 NucliSENS EasyMAG）提取方法的病毒 DNA 回收率。比较显示，自动提取的病毒 DNA 回收率更高（血浆中为 61-76%，尿液中为 52-65%），而手动方法（血浆中为 49-52%，尿液中为 33-56%）较低。使用包含 BKV 全长基因组的质粒的系列稀释液构建外部标准曲线来进行病毒载量的定量。商用定量 BKV 标准与质粒标准具有良好的相关性。该检测方法的重现性基于扩增产物的 Ct 值以及样品中每毫升的 BK 拷贝数来确定。该检测方法在 7 个对数范围内（10 到 1×10(7)拷贝/反应）呈线性，与最接近的多瘤病毒 JCV 以及可能在免疫功能低下患者中发现的其他病毒没有交叉反应，并且与人类基因组 DNA 没有交叉反应。该检测方法在血浆和尿液中的检测限均为 300 拷贝/毫升，使用 ZeptoMetrix 的 NATtrol BK 病毒线性面板，定量限为 1000 拷贝/毫升。该实时 PCR 检测方法为定量检测血浆和尿液样本中的 BKV 提供了一种可靠和敏感的方法。
