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KMID : 0545120100200101463
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Journal of Microbiology and Biotechnology
2010 Volume.20 No. 10 p.1463 ~ p.1470
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Quantitative Detection of Residual E. coli Host Cell DNA by Real-Time PCR
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Lee Dong-Hyuck
Bae Jung-Eun
Lee Jung-Hee
Shin Jeong-Sup
Kim In-Seop
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Abstract
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E. coli has been widely used as a host system to manufacture recombinant proteins for human therapeutic use. Among impurities to be eliminated during the downstream process, residual host cell DNA is a major interest for safety. Residual E. coli host cell DNA in the final products are usually determined using conventional slot blot hybridization assay or total DNA Threshold assay, although these methods are time consuming, expensive, and relatively insensitive. Therefore a sensitive real-time PCR assay for specific detection of residual E. coli DNA was developed and compared with slot blot hybridization assay and Threshold assay to validate the overall capability of these methods. Specific primer pair for amplification of the E. coli 16S rRNA gene was selected to improve the sensitivity, and E. coli host cell DNA was quantified by use of SYBR Green 1. The detection limit of real-time PCR assay in the optimized condition was calculated to be 0.042 pg genomic DNA, which is much higher than those of slot blot hybridization assay and Threshold assay of which detection limit were 2.42 and 3.73 pg genomic DNA, respectively. The real-time PCR assay was validated to be more reproducible, accurate, and precise than slot blot hybridization assay and Threshold assay. The real-time PCR assay may be a useful tool for quantitative detection and clearance validation of residual E. coli DNA during the manufacturing process for recombinant therapeutics.
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KEYWORD
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E. coli host cell DNA, real-time PCR, slot blot hybridization assay, total DNA Threshold assay
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