Detection of replication-competent adenoviruses spiked into recombinant adenovirus vector products by infectivity PCR.
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.
Ishii-Watabe
Akiko
A
Division of Biological Chemistry and Biologicals, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, Japan. watabe@nihs.go.jp
Uchida
Eriko
E
Iwata
Akiko
A
Nagata
Ryuji
R
Satoh
Kouei
K
Fan
Kejun
K
Murata
Mitsuhiro
M
Mizuguchi
Hiroyuki
H
Kawasaki
Nana
N
Kawanishi
Toru
T
Yamaguchi
Teruhide
T
Hayakawa
Takao
T
D000256:Adenoviridae / Q000235:genetics / Q000302:isolation & purification; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction