两种定量巨细胞病毒聚合酶链反应检测方法（Cobas Amplicor CMV Monitor和TaqMan检测法）与pp65抗原血症检测法在测定器官移植患者外周血病毒载量中的比较。

Comparison of two quantitative CMV PCR tests, Cobas Amplicor CMV Monitor and TaqMan assay, and pp65-antigenemia assay in the determination of viral loads from peripheral blood of organ transplant patients.

作者信息

Piiparinen H, Höckerstedt K, Grönhagen-Riska C, Lautenschlager I

机构信息

Department of Virology, Helsinki University Central Hospital and University of Helsinki, Haartmaninkatu 3, FIN-00290 Helsinki, Finland.

出版信息

J Clin Virol. 2004 Jul;30(3):258-66. doi: 10.1016/j.jcv.2003.12.010.

DOI:10.1016/j.jcv.2003.12.010

PMID:15135746

Abstract

BACKGROUND

Quantitative PCR assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. However, usually these tests are still quite time-consuming and labor-intensive which diminishes their utility of these tests in routine diagnostic laboratories.

OBJECTIVES

The objective of this study was to develop a quantitative CMV PCR test which is time-saving and easy to perform for the detection and monitoring of CMV infection of transplant patients.

STUDY DESIGN

The quantitative real time CMV PCR assay using TaqMan chemistry and an automated sample preparation system, MagNA Pure LC, was developed. The designed quantitative CMV test was compared to commercial quantitative PCR test, Cobas Amplicor Monitor, in the determination of CMV DNA loads in plasma samples of liver and kidney transplant patients. The results were also correlated with the CMV pp65-antigenemia test. The clinical material of 270 blood specimens of transplant patients were tested using these two PCR methods and pp65-antigenemia test in parallel. Plasma samples were used for PCR assays and leucocytes for the antigenemia test.

RESULTS

The TaqMan assay described was easy to perform, it was rapid (3-4 h) and hands-on time needed for performing the test was short. The detection limit of the assay was 250 copies/ml (cps/ml) plasma and the linear range up to 25,000,000 cps/ml. TaqMan assay was the most sensitive test detecting 92% of the CMV positive findings. Cobas Monitor detected 80% and pp65 test 88% of the positive findings. The correlations between TaqMan and antigenemia assays, and between Cobas Amplicor and antigenemia were statistically significant and high, R = 0.84 (P < 0.0001) and R = 0.80 (P < 0.0001), respectively. Also correlation between two PCR tests was statistically significant (R = 0.64, P < 0.0001). Of the 27 patient studied, 19 demonstrated CMV antigenemia and DNAemia in their blood during the post transplant monitoring. Thirteen of these patients developed a symptomatic CMV infection and were treated with ganciclovir. The peak viral loads of symptomatic patients were statistically higher by all three methods than those of asymptomatic patients.

CONCLUSIONS

The developed real time TaqMan assay was rapid and easily performed and could be the best alternative for the diagnosis of CMV infection and monitoring of liver and kidney transplant patients.

摘要

背景

定量聚合酶链反应（PCR）检测已成为移植患者巨细胞病毒（CMV）感染期间病毒载量测定中最常用的方法。然而，通常这些检测仍然相当耗时且 labor-intensive，这降低了它们在常规诊断实验室中的实用性。

目的

本研究的目的是开发一种定量 CMV PCR 检测方法，该方法省时且易于操作，用于检测和监测移植患者的 CMV 感染。

研究设计

开发了使用 TaqMan 化学和自动化样品制备系统 MagNA Pure LC 的定量实时 CMV PCR 检测方法。在测定肝移植和肾移植患者血浆样本中的 CMV DNA 载量时，将设计的定量 CMV 检测方法与商业定量 PCR 检测方法 Cobas Amplicor Monitor 进行比较。结果还与 CMV pp65 抗原血症检测相关。使用这两种 PCR 方法和 pp65 抗原血症检测对 270 份移植患者的血液标本临床材料进行了平行检测。血浆样本用于 PCR 检测，白细胞用于抗原血症检测。

结果

所述的 TaqMan 检测方法易于操作，速度快（3 - 4 小时），且进行检测所需的实际操作时间短。该检测方法的检测限为 250 拷贝/毫升（cps/ml）血浆，线性范围高达 25,000,000 cps/ml。TaqMan 检测是最灵敏的检测方法，检测出 92%的 CMV 阳性结果。Cobas Monitor 检测出 80%，pp65 检测检测出 88%的阳性结果。TaqMan 检测与抗原血症检测之间以及 Cobas Amplicor 检测与抗原血症检测之间的相关性具有统计学意义且高度相关，R 分别为 0.84（P < 0.0001）和 0.80（P < 0.0001）。两种 PCR 检测方法之间的相关性也具有统计学意义（R = 0.64，P < 0.0001）。在研究的 27 名患者中，19 名在移植后监测期间血液中出现了 CMV 抗原血症和病毒血症。其中 13 名患者发生了有症状的 CMV 感染并接受了更昔洛韦治疗。所有三种方法检测到的有症状患者的病毒载量峰值在统计学上均高于无症状患者。