Detection of replication-competent adenoviruses spiked into recombinant adenovirus vector products by infectivity PCR
Detection of replication-competent adenoviruses spiked into recombinant adenovirus vector products by infectivity PCR
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DOI：
10.1016/j.ymthe.2003.09.001
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发表时间：
2003-12-01
影响因子：
12.4
通讯作者：
Hayakawa, T
中科院分区：
文献类型：
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作者：
Ishii-Watabe, A;Uchida, E;Hayakawa, T
可复制性腺病毒(RCA)在临床上大量的腺病毒载体中的存在引起了各种安全问题。要检测腺病毒载体产物中的RCA，一般首选细胞培养/细胞病变效应(CPE)方法。然而，用这种方法很难清楚、定量地评价RCA的数量。此外，细胞培养/CPE方法需要大规模的细胞培养和相当长的时间。为了建立一种更灵敏、更快速地检测RCA的方法，我们发展了传染性PCR，这是一种感染性检测和定量PCR相结合的混合方法。该方法可以利用针对E1DNA设计的引物和探针，通过实时定量聚合酶链式反应对RCA进行定量。通过感染性聚合酶链式反应，可在10(9)个腺病毒载体颗粒中检测到1pfu的RCA。相反，在腺病毒载体的10(9)个颗粒中加入10(4)pfu的RCA，在感染细胞中观察到CPE。玻璃微珠法适合于快速提取RCA感染细胞的DNA。结果表明，感染性聚合酶链式反应结合玻璃微球DNA提取法可用于腺病毒载体产物中RCA的检测。
The presence of replication-competent adenovirus (RCA) in clinical lots of adenovirus vectors raises a variety of safety concerns. To detect RCA in adenovirus vector products, the cell culture/ cytopathic effect (CPE) method has generally been preferred. However, it is difficult to evaluate the amount of RCA clearly and quantitatively by this method. In addition, the cell culture/CPE method requires large-scale cell culturing and a substantial amount of time. For the purpose of establishing a method to detect RCA more sensitively and rapidly, we developed the infectivity PCR, a hybrid method that combines the infectivity assay and quantitative PCR. This method allows RCA to be quantified by real-time quantitative PCR using primers and a probe designed for E1 DNA. By infectivity PCR, 1 pfu of RCA spiked into 10(9) particles of adenovirus vectors could be detected. In contrast, CPE was observed in the cells infected with 10(4) pfu of RCA spiked into 10(9) particles of adenovirus vectors. The glass-beads method was suitable for extracting DNA rapidly from the RCA-infected cells. These results showed that infectivity PCR combined with the glass-beads-based DNA extraction method was useful for the detection of RCA in adenovirus vector products.