Specific detection of residual CHO host cell DNA by real-time PCR
Specific detection of residual CHO host cell DNA by real-time PCR
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DOI：
10.1016/j.biologicals.2006.09.001
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发表时间：
2007-06-01
期刊：
影响因子：
1.7
通讯作者：
Nissom, Peter Morin
中科院分区：
文献类型：
--
作者：
Nissom, Peter Morin
中国仓鼠卵巢细胞已被广泛用于生产用于人类治疗用途的重组蛋白。建立了一种灵敏的实时荧光定量聚合酶链反应（PCR）检测中国仓鼠（Cricetulus griseus）DNA残留量的方法。该测定是合理负担得起的，并且可以适用于使用96孔格式的高通量筛选。设计实时PCR引物以从仓鼠DNA扩增基因组片段的150bp区域。使用来自小鼠成纤维细胞、人肾脏和仓鼠卵巢细胞系的基因组DNA作为模板，在实时PCR反应中评价探针的特异性。在来自仓鼠细胞系CHO DG44的基因组DNA上比较实时PCR的灵敏度。这些引物可用于实时PCR反应，以检测纯化蛋白质样品中污染仓鼠DNA的存在，灵敏度低至300 fg基因组DNA。(c)2006年，国际生物制品协会。由爱思唯尔有限公司出版。保留所有权利。
Chinese hamster ovary cells have been widely used to manufacture recombinant proteins for human therapeutic use. A sensitive quantitative real-time polymerase chain reaction assay for the detection of residual Chinese hamster (Cricetulus griseus) DNA is presented in this paper. The assay is reasonably affordable and can be adapted for high-throughput screening using 96-well format. Real-time PCR primers were designed to amplify a 150 bp region of a genomic fragment from hamster DNA. The specificity of the probe was evaluated in real-time PCR reactions using genomic DNA from mouse fibroblast, human kidney and hamster ovary cell lines as template. Sensitivity of real-time PCR was compared on genomic DNA from hamster cell line CHO DG44. These primers can be used in real-time PCR reactions to detect presence of contaminating hamster DNA in purified protein samples down to sensitivity of 300 fg genomic DNA. (c) 2006 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.