三种实时 PCR 方法用于定量检测多瘤病毒 BK 的比较

Comparison of three real-time PCR for the quantification of polyomavirus BK.

机构信息

Department of Pathology and Laboratory Medicine, Albany Medical Center Hospital, Albany, NY, USA.

出版信息

J Clin Virol. 2013 Apr;56(4):354-9. doi: 10.1016/j.jcv.2012.12.003. Epub 2012 Dec 28.

DOI:10.1016/j.jcv.2012.12.003

PMID:23273665

Abstract

BACKGROUND

Reactivation of latent polyomavirus BK is associated with nephropathy (PVAN) after renal transplantation. BK viral load determinations are a highly sensitive and specific method for predicting risk for PVAN.

OBJECTIVES AND STUDY DESIGN

The performance of three real-time PCR for BKV DNA quantification (MultiCode(®)-RTx BK virus ASR [MC-RTx], MGB-Alert BKV ASR [MGB] and a laboratory developed assay [LDA]) were evaluated against a conventional PCR (test of record, TOR) in terms of linearity, dynamic range, and accuracy.

RESULTS

The LOD (log(10) copies/ml) were 2.0, 2.0 and 3.0 for MC-RTx, MGB and LDA, respectively with a commercial plasma panel and 2.0, 2.6 and 3.5 with a urine panel. These assays demonstrated excellent linearity (r(2) = 1.0) and reproducibility (CV range = 0.7-20.4%, 0.9-13.2%, and 0.5-13%, respectively). In an analysis of 100 clinical specimens, all 76 samples defined as true positive for BKV DNA (positive by two or more methods or a recent history of positivity) were detected with MC-RTx, while only 64 were detected with MGB and 55 were detected with LDA. BKV DNA was not detected by any method in the true negative specimens. Based on these results, the sensitivities were 100% for MC-RTx, 84% for MGB and 72% for LDA. The greatest linear correlation with the mean concentration was observed with MC-RTx (r(2) = 0.96) with two samples (3%) with greater than 0.5 log(10) variance in quantification versus seven (11%) with MGB and ten (18%) with LDA.

CONCLUSIONS

These real-time assays for BKV load demonstrated excellent performance characteristics, with the MC-RTx demonstrating the greatest sensitivity.

摘要

背景

潜伏的多瘤病毒 BK 的再激活与肾移植后的肾病（PVAN）有关。BK 病毒载量的测定是预测 PVAN 风险的一种高度敏感和特异的方法。

目的和研究设计

我们评估了三种实时 PCR 用于 BKV DNA 定量的性能（MultiCode®-RTx BK 病毒 ASR[MC-RTx]、MGB-Alert BKV ASR[MGB]和实验室开发的检测方法 [LDA]），与常规 PCR（记录测试，TOR）相比，在线性、动态范围和准确性方面进行了评估。

结果

用商业血浆板，LOD（log(10)拷贝/ml）分别为 2.0、2.0 和 3.0，用尿液板分别为 2.0、2.6 和 3.5。这些检测方法表现出极好的线性（r(2)=1.0）和重复性（CV 范围分别为 0.7-20.4%、0.9-13.2%和 0.5-13%）。在对 100 个临床标本的分析中，用 MC-RTx 检测到所有 76 个样本（用两种或更多方法或近期阳性史定义为 BKV DNA 阳性），而 MGB 仅检测到 64 个，LDA 仅检测到 55 个。在真正的阴性标本中，没有任何方法检测到 BKV DNA。基于这些结果，MC-RTx 的敏感性为 100%，MGB 为 84%，LDA 为 72%。与平均浓度的线性相关性最大的是 MC-RTx（r(2)=0.96），与 MGB 相比，有两个样本（3%）的定量差异大于 0.5 log(10)，而与 LDA 相比，有十个样本（18%）的定量差异大于 0.5 log(10)。