一种通过微载体细胞培养和定量PCR相结合来检测具有复制能力的腺病毒的快速灵敏检测方法。

A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR.

作者信息

Schalk Johanna A C, de Vries Claudette G J C A, Orzechowski Tom J H, Rots Marianne G

机构信息

National Institute for Public Health and the Environment, Centre for Biological Medicines and Medical Technology, The Netherlands.

出版信息

J Virol Methods. 2007 Nov;145(2):89-95. doi: 10.1016/j.jviromet.2007.05.010. Epub 2007 Jun 22.

DOI:10.1016/j.jviromet.2007.05.010

PMID:17588680

Abstract

The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by E1-specific quantitative PCR. The detection limit of this assay is 3 RCAs in 1 x 10(10) vector particles per 70 ml of microcarrier cell culture. The main advantage of the combination of cell culture and PCR detection is that replicated virus can be detected long before cytopathic effects become visible and therefore, it is much faster than conventional cell culture-based assays. This assay was validated by spiking replication-incompetent adenoviral vectors with wild-type adenovirus serotype 5 (wt Ad5) as a positive control for RCA. It was found that the replication of wt Ad5 is hampered above a vector particle per cell ratio of 50. However, if microcarrier beads are used, many cells can be grown in a small suspension culture and consequently a large number of vector particles can be tested for contamination with RCA.

摘要

本文描述了一种用于检测具有复制能力的腺病毒（RCA）的快速灵敏检测方法。该RCA检测方法包括在微载体细胞培养系统中，将腺病毒载体在A549细胞上孵育4天的步骤，随后通过E1特异性定量PCR检测扩增的RCA。该检测方法的检测限为每70 ml微载体细胞培养物中，每1×10(10)个载体颗粒中有3个RCA。细胞培养和PCR检测相结合的主要优点是，在细胞病变效应可见之前很久就能检测到复制的病毒，因此，它比传统的基于细胞培养的检测方法快得多。通过用野生型腺病毒血清型5（wt Ad5）作为RCA的阳性对照，对无复制能力的腺病毒载体进行加样验证了该检测方法。结果发现，当每个细胞的载体颗粒比例高于50时，wt Ad5的复制受到阻碍。然而，如果使用微载体珠，可以在小体积悬浮培养中培养许多细胞，因此可以检测大量载体颗粒是否被RCA污染。