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首页> 外文期刊>Journal of microbiology and biotechnology >Quantitative detection of residual E. coli host cell DNA by real-time PCR
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Quantitative detection of residual E. coli host cell DNA by real-time PCR

机译:通过实时PCR定量检测残留的大肠杆菌宿主细胞DNA

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摘要

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.
机译:长期以来,大肠杆菌一直广泛用作宿主系统,用于生产旨在用于人类治疗的重组蛋白。当考虑在下游过程中消除杂质时,残留的宿主细胞DNA是主要的安全隐患。通常使用常规的狭缝印迹杂交测定法或总DNA阈值测定法确定最终产物中残留的大肠杆菌宿主细胞DNA的存在。但是,前一种方法和后一种方法都是费时,昂贵且相对不敏感的。因此,这项研究尝试开发一种更灵敏的实时PCR分析方法,用于残留大肠杆菌DNA的特异性检测。然后将该新方法与狭缝印迹杂交测定法和总DNA阈值测定法进行比较,以确定其有效性和整体能力。新方法涉及选择特定的引物对以扩增大肠杆菌16S rRNA基因,以提高灵敏度,而大肠杆菌宿主细胞DNA定量则通过使用SYBR Green I进行。在这些优化条件下,实时荧光定量PCR的结果被计算为0.042 pg基因组DNA,这比狭缝印迹杂交分析和总DNA阈值分析(检测限分别为2.42和3.73 pg)要高得多基因组DNA。因此,可以说实时PCR检测比狭缝印迹杂交检测或总DNA阈值检测更可重现,更准确和更精确。因此,实时PCR分析可能是一种有前途的新工具,用于在重组治疗剂的生产过程中对残留的大肠杆菌宿主细胞DNA进行定量检测和清除验证。

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