Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay
Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay
复制标题
DOI：
10.1016/s0166-0934(03)00129-0
复制
发表时间：
2003-06-30
影响因子：
3.1
通讯作者：
Niedrig, M
中科院分区：
文献类型：
--
作者：
Bae, HG;Nitsche, A;Niedrig, M
黄热病病毒的定量通常是通过使用敏感细胞培养含有病毒的样本来进行的。对产生的斑块的计数提供了样本中存在的感染颗粒数量的标记。根据所使用的黄热病病毒株的不同，这种化验通常需要长达5天的时间才能得到结果，并且必须在L2或U实验室条件下进行。对于黄热病病毒感染的临床诊断，基于细胞培养的方法耗时太长，实际意义有限。近年来，由于其相当高的灵敏度，聚合酶链式反应已成为一种很有前途的病毒检测方法。然而，尽管聚合酶链式反应可以检测病毒特异性核酸，但它不允许就检测到的病毒的感染潜力得出结论。然而，为了诊断的目的，快速、特异和敏感的病毒聚合酶链式反应是可取的。因此，建立了两种独立的黄热病病毒特异性实时定量聚合酶链式反应方法，并将病毒RNA载量与传统的空斑分析结果进行了比较。黄热病病毒基因组与感染颗粒的估计比例在1000：1到5000：1之间；两种方法显示出类似的精确度为45%。实时定量聚合酶链式反应测定的基因组数目与配对样本中相应的斑块数目之间存在显著的相关性，皮尔逊相关系数r=0.88(P<；0.0001)。(C)2003 Elsevier Science B.V.保留所有权利。
Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or U laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000: 1 and 5000: 1; both approaches displayed a comparable precision of < 45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r = 0.88 (P < 0.0001). (C) 2003 Elsevier Science B.V. All rights reserved.