Evaluation of a quantitative real-time PCR for the detection of respiratory syncytial virus in pulmonary diseases
Evaluation of a quantitative real-time PCR for the detection of respiratory syncytial virus in pulmonary diseases
复制标题
DOI：
10.1183/09031936.03.00088102
复制
发表时间：
2003-06-01
影响因子：
24.3
通讯作者：
Bufe, A
中科院分区：
文献类型：
--
作者：
Borg, I;Rohde, G;Bufe, A
已知呼吸道合胞病毒（RSV）可引起幼儿急性下呼吸道感染（ARI），并参与成人慢性阻塞性肺疾病（COPD）的加重。RSV在稳定期COPD中的作用以及不同呼吸道疾病中的病毒载量至今尚未研究，本文作者建立并评价了一种针对RSV A亚群的定量TaqMan（R）实时聚合酶链反应检测方法。使用对照质粒实现用于测定病毒载量输入的绝对定量。该检测方法的定量范围大于6个对数，检测限< 10个RSV拷贝/反应混合物，灵敏度是常规巢式聚合酶链反应检测方法的30倍。批间标准差平均为1.3，变异系数平均为4.7%。比较了ARI婴儿（n = 62）和COPD老年人（n = 125）的临床标本的病毒载量。在ARI研究组中共发现47%的RSV阳性样本，在COPD研究组中为28%。发现两个研究组的病毒载量显著不同。在ARI研究组中，病毒载量几乎增加了2000倍，表明该组中存在急性感染，而COPD组中存在既往或潜伏感染。呼吸道合胞病毒-A特异性TaqMan（R）实时聚合酶链反应测定法是测定临床样本中病毒载量的一种灵敏和快速的方法。它使有关呼吸道合胞病毒参与急性下呼吸道感染和慢性阻塞性肺疾病的区别声明得以实现。
Respiratory syncytial virus (RSV) is known to cause acute lower respiratory tract infections (ARI) in young children and is involved in exacerbation of chronic obstructive pulmonary disease (COPD) in adults. The role of RSV in stable COPD and the viral load in different respiratory diseases has not been investigated to date.The present authors established and evaluated a quantitative TaqMan(R) real-time polymerase chain reaction assay specific for RSV subgroup A. Absolute quantification for the determination of viral load input was achieved using a control plasmid. The assay allowed for a quantification over a > 6-log range and a detection limit of < 10 RSV copies per reaction mixture.The assay was 30 times more sensitive than conventional nested polymerase chain reaction assays. Interassay SD was 1.3 and coefficient of variation 4.7% on average. Clinical specimens from infants with ARI (n = 62) and elderly adults with COPD (n = 125) were compared for viral loads. A total of 47%, RSV-positive samples were found in the ARI study group and 28% in the COPD study group. The viral load of both study groups was found to differ significantly. In the ARI study group the viral load was increased almost 2000-fold, suggesting acute infection in this group and former or latent infection in the COPD group.Respiratory syncytial virus-A specific TaqMan(R) real-time polymerase chain reaction assay is a sensitive and rapid method for the determination of viral load in clinical samples. It enables differential statements concerning the involvement of respiratory syncytial virus in acute lower respiratory tract infections and chronic obstructive pulmonary disease to be achieved.