Delelis Olivier, Brussel Audrey, Sonigo Pierre
Département des Maladies Infectieuses, Institut Cochin, INSERM 1567, CNRS UMR 81041, Université René Descartes, Paris, France.
Methods Mol Biol. 2005;304:155-70. doi: 10.1385/1-59259-907-9:155.
Integration is described as a key step in viral replication of all retroviruses. A sensitive and quantitative measure of an integrated molecule is a good way to examine the importance of the integration step and to evaluate efficiency of retroviral vectors for gene transfer or anti-integrase drugs. Here, we report a sensitive and quantitative real-time polymerase chain reaction (PCR) technique to measure integrated viral DNA in human cells during a foamy virus (HFV) infection. This technique is based on two steps of PCR. The first round amplifies Alu-LTR (long terminal repeat) sequences resulting from viral integration. The second round of PCR is performed to quantify these events of integration. Quantification is monitored by the comparison of the amplification curve of the sample against a standard scale constituted of viral DNA from chronically infected cells. Sensitivity of this technique allows us to detect as few as 25 copies of HFV-integrated DNA in 50,000 cells.
整合被描述为所有逆转录病毒病毒复制中的关键步骤。对整合分子进行灵敏且定量的检测是检验整合步骤的重要性以及评估逆转录病毒载体用于基因转移或抗整合酶药物效率的良好方法。在此，我们报告一种灵敏且定量的实时聚合酶链反应（PCR）技术，用于在泡沫病毒（HFV）感染期间测量人细胞中整合的病毒DNA。该技术基于两步PCR。第一轮扩增由病毒整合产生的Alu-LTR（长末端重复序列）序列。进行第二轮PCR以量化这些整合事件。通过将样品的扩增曲线与由慢性感染细胞的病毒DNA构成的标准曲线进行比较来监测定量。该技术的灵敏度使我们能够在50,000个细胞中检测到低至25个拷贝的HFV整合DNA。
