Merlino Chiara, Bergallo Massimiliano, Gribaudo Giorgio, Gregori Gabriella, Paolo Segoloni Giuseppe, Giacchino Franca, Ponzi Alessandro Negro, Cavallo Rossana
Department of Public Health and Microbiology, Virology Unit, University of Turin, Via Santena 9-10126, Turin, Italy.
J Clin Virol. 2003 Dec;28(3):265-74. doi: 10.1016/s1386-6532(03)00012-x.
Several studies have disclosed a correlation between polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients and its quantification in urine and serum is therefore required to assess the role of BKV infection in nephropathy.
This paper describes a urine and serum BKV-DNA quantification protocol devised to evaluate the viral load.
Screening of samples containing > or =10(3)/ml viral genome copies by a semi-quantitative polymerase chain reaction (PCR) assay is followed by precise quantification of the samples containing a high number of viral genomes in a quantitative-competitive (QC)-PCR assay. Generation of the competitor construct relied on the different sizes of wild-type and competitor amplicons.
Screening by semi-quantitative PCR selects samples with a high number of viral genomes for use in the more labor-intensive and -expensive QC-PCR assay and thus provides a handy means for quantitative DNA analysis of large numbers of samples. The results obtained in BKV-DNA quantification in urine and serum samples from 51 renal transplant recipients (22 on treatment with tacrolimus (FK506) and 29 on cyclosporine A (Cy A)) are interesting: BKV-DNA findings (43.1%) in urine samples are in agreement with the BKV urinary shedding reported in literature (5-45%). With regard to immunosuppressive treatment, the percentage of activation of the infection (revealed by BKV-DNA detection in urine samples) in the two groups of therapy is similar (40.9% vs 44.8%). The observation that the viral load in urine is dissociated with that of serum suggests that both parameters should be investigated in evaluation of the pathogenetic role of BKV reactivation in renal transplant recipients. Moreover, our BKV-DNA quantification protocol could be used to monitor viral load in urine and serum samples from renal transplant recipients so as to detect those at risk of nephropathy and monitor their response to immunosuppression reduction therapy if it occurs.
多项研究揭示了多瘤病毒BK（BKV）与肾移植受者间质性肾炎之间的相关性，因此需要对尿液和血清中的BKV进行定量检测，以评估BKV感染在肾病中的作用。
本文描述了一种用于评估病毒载量的尿液和血清BKV-DNA定量检测方法。
通过半定量聚合酶链反应（PCR）检测筛选出病毒基因组拷贝数≥10³/ml的样本，然后采用定量竞争（QC）-PCR检测对病毒基因组数量较多的样本进行精确定量。竞争构建体的产生依赖于野生型和竞争扩增子的不同大小。
通过半定量PCR筛选出病毒基因组数量较多的样本，用于更耗时且昂贵的QC-PCR检测，从而为大量样本的DNA定量分析提供了一种便捷方法。对51例肾移植受者（22例接受他克莫司（FK506）治疗，29例接受环孢素A（Cy A）治疗）的尿液和血清样本进行BKV-DNA定量检测，结果有趣：尿液样本中的BKV-DNA检测结果（43.1%）与文献报道的BKV尿排泄率（5%-45%）一致。关于免疫抑制治疗，两组治疗中感染激活的百分比（通过尿液样本中的BKV-DNA检测显示）相似（40.9%对44.8%）。尿液中病毒载量与血清中病毒载量分离的观察结果表明，在评估BKV再激活在肾移植受者发病机制中的作用时，应同时研究这两个参数。此外，我们的BKV-DNA定量检测方法可用于监测肾移植受者尿液和血清样本中的病毒载量，以检测有肾病风险的患者，并在其接受免疫抑制减量治疗时监测其反应。
