Quantitative Detection of Residual E. coli Host Cell DNA by Real-Time PCR

• Published in: Journal of microbiology and biotechnology Vol. 20; no. 10; pp. 1463 - 1470
• Main Authors: Lee, D.H., Hannam University, Daejeon, Republic of Korea, Bae, J.E., Hannam University, Daejeon, Republic of Korea, Lee, J.H., Hannam University, Daejeon, Republic of Korea, Shin, J.S., Green Cross Corp., Cheongwon, Republic of Korea, Kim, I.S., Hannam University, Daejeon, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.10.2010; 한국미생물·생명공학회
• Subjects: Biological and medical sciences; Biotechnology; DNA Primers - genetics; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; E. coli host cell DNA; ESCHERICHIA COLI; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Polymerase Chain Reaction - methods; real-time PCR; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; slot blot hybridization assay; total DNA Threshold assay; 생물학; Polymerase chain reaction; Molecular hybridization; DNA; E. coli host cell DNA; total DNA Threshold assay; real-time PCR; Detection; Real time; slot blot hybridization assay; Quantitative analysis
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.1004.04035

E. coli has long been widely used as a host system for the manufacture of recombinant proteins intended for human therapeutic use. When considering the impurities to be eliminated during the downstream process, residual host cell DNA is a major safety concern. The presence of residual E. coli host cell DNA in the final products is typically determined using a conventional slot blot hybridization assay or total DNA Threshold assay. However, both the former and latter methods are time consuming, expensive, and relatively insensitive. This study thus attempted to develop a more sensitive real-time PCR assay for the specific detection of residual E. coli DNA. This novel method was then compared with the slot blot hybridization assay and total DNA Threshold assay in order to determine its effectiveness and overall capabilities. The novel approach involved the selection of a specific primer pair for amplification of the E. coli 16S rRNA gene in an effort to improve sensitivity, whereas the E. coli host cell DNA quantification took place through the use of SYBR Green I. The detection limit of the real-time PCR assay, under these optimized conditions, was calculated to be 0.042 pg genomic DNA, which was much higher than those of both the slot blot hybridization assay and total DNA Threshold assay, where the detection limits were 2.42 and 3.73 pg genomic DNA, respectively. Hence, the real-time PCR assay can be said to be more reproducible, more accurate, and more precise than either the slot blot hybridization assay or total DNA Threshold assay. The real-time PCR assay may thus be a promising new tool for the quantitative detection and clearance validation of residual E. coli host cell DNA during the manufacturing process for recombinant therapeutics.