Tafuro S, Zentilin L, Falaschi A, Giacca M
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Gene Ther. 1996 Aug;3(8):679-84.
A quantitative polymerase chain reaction (PCR) procedure has been developed for rapid retrovirus titration. This procedure, which is based on the simultaneous amplification of the sample with known amounts of a competitor DNA fragment (competitive PCR), was used for the quantification of viral RNA genomes in retrovirus-producing cell clone supernatants and of proviral DNA molecules formed at 24 h after infection of different reference cell lines. The results obtained from the analysis of several samples indicated that proviral DNA quantification is in complete agreement with the number of selectable colonies in a standard colony assay. Conversely, the number of viral RNA genomes in the producer cell clone supernatants is a poor predictor of the actual efficiency of infection. Repeated competitive PCR experiments for provirus copy number determination at different times after transduction indicated that the number of proviral DNA molecules remains stable over time, suggesting stable integration into the host genome. The developed procedure is rapid and simple, is applicable to retroviral constructs not containing a selectable gene and can be used to directly measure the efficiency of infection of any target cell type, thus overcoming the problem of the dependency of retroviral titer determination on the rate of expression of a selectable gene and on the efficiency of colony formation of a reference cell line.
已开发出一种用于快速逆转录病毒滴定的定量聚合酶链反应（PCR）方法。该方法基于用已知量的竞争DNA片段同时扩增样品（竞争性PCR），用于定量逆转录病毒产生细胞克隆上清液中的病毒RNA基因组以及不同参考细胞系感染24小时后形成的前病毒DNA分子。对多个样品分析所得结果表明，前病毒DNA定量与标准集落测定中的可选择集落数完全一致。相反，产生细胞克隆上清液中病毒RNA基因组的数量并不能很好地预测实际感染效率。在转导后不同时间重复进行用于前病毒拷贝数测定的竞争性PCR实验表明，前病毒DNA分子的数量随时间保持稳定，这表明其稳定整合到宿主基因组中。所开发的方法快速且简单，适用于不含可选择基因的逆转录病毒构建体，可用于直接测量任何靶细胞类型的感染效率，从而克服了逆转录病毒滴度测定依赖于可选择基因表达速率和参考细胞系集落形成效率的问题。
