Detection of residual E. coli host cell DNA by 23S ribosomal RNA gene-targeted quantitative polymerase chain reactions
| Autor: | Qian Zhang, Mu Yang, Linsong Zhang, Peizi He, Zhangjie Gu, Guodi Liu, Yingjiao Pan, Jibin Liu, Xingbing Cui, Guoping Liu, Xiaoli Tian, Xiang Li, Dehua Li |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
DNA
Bacterial Clinical Biochemistry Pharmaceutical Science medicine.disease_cause 01 natural sciences Polymerase Chain Reaction Analytical Chemistry chemistry.chemical_compound Plasmid 23S ribosomal RNA RNA Ribosomal 16S Drug Discovery medicine Escherichia coli Gene Spectroscopy Polymerase biology 010405 organic chemistry 010401 analytical chemistry Genes rRNA DNA 0104 chemical sciences RNA Ribosomal 23S Biochemistry chemistry biology.protein DNA fragmentation Primer (molecular biology) |
| Zdroj: | Journal of pharmaceutical and biomedical analysis. 198 |
| ISSN: | 1873-264X |
| Popis: | Among the many systems available for heterologous protein production gram-negative bacterium Escherichia coli (E. coli) has long been widely used because of its ability to grow rapidly with a high density on inexpensive substrates. The use of E. coli as the host system has many regulatory issues, one of which is the residual host cell DNA. Residual DNA carried by biological products may lead to carcinogenicity and immunomodulation risks. The World Health Organization (WHO) for the acceptable amounts of residual host cell DNA is less than 10 ng per dose. Therefore, it is important to keep an extremely low level of residual host DNA in the biological products derived from E. coli. In this study, we designed primer/probe sets targeting E. coli 23S ribosomal RNA gene to quantify the residual DNA of E. coli by quantitative polymerase chain reactions (qPCR). Result showed that this primer/probe has high species specificity. The limit of detection (LOD) in this method is 0.01 pg/μl and this allowed for detection of residual host DNA of much lower concentrations. We assessed accuracy by calculating the recovery (92.1∼140.1 %) of the spiked DNA in plasmids which were produced from E. coli. We also checked intra-assay precision (9.8∼15.1 %) and inter-assay precision (10.9∼18.3 %) by repeatedly measuring the four different concentration standards. In addition, the robustness assay was performed by generating standard curve using short length E. coli DNA. The result showed that appropriate degree of DNA fragmentation will not affect tests. These validation studies demonstrated that our method has excellent specificity, linearity, accuracy, precision and robustness. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect