实时 RT-PCR 检测细胞和细胞系中的逆转录病毒污染。
Real-time RT-PCR detection of retroviral contaminations of cells and cell lines.
机构信息
Molecular Biotechnology, German Research Centre of Biotechnology, GBF, Braunschweig, Germany.
出版信息
Cytotechnology. 2002 Jan;38(1-3):147-53. doi: 10.1023/A:1021126703683.
We have developed a fast and sensitive on-line detection method for retroviruses using the PCR technology. The assay utilizes the endogenous reverse transcriptase activity in retroviral particles. In the presence of active reverse transcriptase, bacteriophage MS2 RNA is transcribed into cDNA and is subsequently amplified in a SYBR-Green-type LightCyclertrade mark reaction. The method allows a qualitative and quantitative monitoring of RT-activity, is several orders of magnitude more sensitive than a standard RT assay and has a time requirement of 2.5 hours from harvest to result. The methodis useful for monitoring of cells and cell-derived products, viral vectors and recombinant proteins for the presence ofreplication-competent retroviruses (RCRs).
我们利用 PCR 技术开发了一种快速灵敏的逆转录病毒在线检测方法。该检测方法利用了逆转录病毒颗粒中内源性逆转录酶的活性。在有活性的逆转录酶存在的情况下，噬菌体 MS2 RNA 被转录成 cDNA，随后在 SYBR-Green 型 LightCyclertrade mark 反应中进行扩增。该方法可以定性和定量监测 RT 活性，比标准 RT 检测方法灵敏几个数量级，从收获到结果的时间要求为 2.5 小时。该方法可用于监测细胞和细胞来源的产品、病毒载体和重组蛋白中是否存在复制型逆转录病毒 (RCRs)。
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